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Molecular systematics and taxonomy of Sporacestra and relatives
(Ramalinaceae, Ascomycota)
Malin Stapnes Dahl
Master of Science thesis
Natural History Museum Department of Biosciences
University of Oslo 2017
II
III
Molecular systematics and taxonomy of
Sporacestra and relatives (Ramalinaceae,
Ascomycota)
Malin Stapnes Dahl
+47 915 63 123
Supervisors
Einar Timdal
Mika Bendiksby
Sonja Kistenich
ISOP group
Natural History Museum
Department of Biosciences
University of Oslo
2017
IV
© Malin Stapnes Dahl
2017
Molecular systematics and taxonomy of Sporacestra and relatives (Ramalinaceae,
Ascomycota)
Author: Malin Stapnes Dahl
http://www.duo.uio.no/
Print: Reprosentralen, University of Oslo
V
Abstract
Tropical members of the Ramalinaceae remain poorly studied. In this master thesis, I have
addressed the phylogenetic relationship within a clade of genera occurring in the Tropics and
Subtropics, including members of Bacidia, Bacidiopsora, Phyllopsora and a resurrected
Sporacestra. A phylogeny based on a nuclear (ITS) and a mitochondrial (SSU) DNA-region
is presented. Bayesian and parsimony phylogenetic analyses on a total of 57 taxa, supports the
monophyly of Sporacestra. Bacidia s. lat. was recovered as polyphyletic. Members of
Bacidiopsora and Phyllopsora were nested into a monophyletic Bacidia s. str. Based on the
molecular phylogenetic results, I describe three new species in Sporacestra and lump
Bacidiopsora and one member of Phyllopsora into Bacidia s. str. According to the
phylogenetic hypothesis presented, thallus architecture and secondary chemistry proved be a
poor diagnostic character at the genus level, being present in multiple genera in the
Ramalinaceae. Five new species, Bacidia corallina Stapnes & Timdal, B. kariegae Stapnes,
Sporacestra isidata Stapnes & Timdal, S. longispora Stapnes and S. straminea Stapnes are
described. Phyllopsora borbonica is reduced to synonymy with P. pertexta, and a new
combination of this species, S. pertexta (Nyl.) Stapnes & Timdal is presented. Two new
names are given, namely Bacidia microphylla Stapnes and B. soralifera Stapnes & Timdal. A
key to Sporacestra is provided.
VI
VII
Acknowledgements
First of all, I would like to give a sincere thanks to my research group at Natural History Museum in
Oslo, for all the support, motivation and help throughout this master. This includes my main
supervisor Einar Timdal, and co-supervisors Mika Bendiksby and Sonja Kistenich. I am forever
grateful for all the guidance, discussions, and constructive feedback you have given me these last two
years. You are all a great source of inspiration, and I would never have been able to finish this thesis
without you. I would also like to thank Siri Rui, for all her help, kindness and support. A special
thanks to fellow student and friend Anne Karin Toreskaas. Having you by my side in the field, during
conferences, in courses, and in the lab has made these two years even better.
I would like to thank Karl-Henrik Larsson for applying for the student grant from SIU – Norwegian
Centre for International Cooperation in Education, making the fieldwork in South America possible.
This was an amazing opportunity, not only being able to study my species in the field, but to
experience parts of South America and to meet so many great local researchers!
For the fieldwork in Venezuela, I would like to thank Jesus Hernandez and his students at Universidad
Central de Venezuela. I am grateful for the exchange of knowledge and all your hospitality. Without
your help, fieldwork in Venezuela would not be possible to carry out.
A special thanks to Tatiana B. Gibertoni for organizing research permits in Brazil. For the fieldwork in
Pará, I would like to acknowledge Denis Conrado for all the translations and Selma Ohashi from
UFPA for help during fieldwork. We could never have done this part of the fieldwork without Milton
Cordeiro and Iracema Coimbra Cordeiro at Fazenda Agroecológica São Roque, who let us carry out
our fieldwork on their land.
In São Paolo, I would like to thank Adriana de Mello Gugliotta and Viviana Motato-Vásquez at
Instituto de Botânica, in Jardim Botânico de São Paulo for all the help during the fieldwork in Itatiaia
National park, and for letting us stay in the botanical garden.
Thanks to Pat Wolseley, Marcela Cáceres and all the curators of BM, BORH, BR, ISE, O, UPS, WIS
and the private herbaria of A. Frisch, A. Paukov, S. Pérez-Ortega and M. Seaward for all the borrowed
material.
This thesis would not be possible without the help from the DNA lab at the Natural History Museum
in Oslo. A special thanks to Birgitte Lisbeth Graae Thorbek for all the helpful lab instructions!
Last, but not least, a warm and deep thanks to friends and family for the support they have given me,
especially to Silje Marie Kristiansen for proofreading my thesis.
Malin Stapnes Dahl
Natural History Museum in Oslo, may 30th, 2017
VIII
Table of Contents
1 Introduction ........................................................................................................................ 1
2 Material and methods ......................................................................................................... 9
2.1 Fieldwork ..................................................................................................................... 9
2.2 The specimens ........................................................................................................... 10
2.3 DNA work ................................................................................................................. 10
2.3.1 DNA-extraction .................................................................................................. 10
2.3.2 Polymerase Chain Reaction (PCR) .................................................................... 11
2.3.3 Gel electrophoresis ............................................................................................. 12
2.3.4 Cleaning PCR-products ...................................................................................... 12
2.3.5 Cycle sequencing ................................................................................................ 13
2.3.6 Ethanol precipitation .......................................................................................... 13
2.3.7 DNA sequencing ................................................................................................ 13
2.3.8 Sequence editing and alignment assembly ......................................................... 14
2.3.9 Data assembly .................................................................................................... 14
2.4 Phylogenetic analyses and model selection ............................................................... 15
2.5 Morphological investigations .................................................................................... 15
2.6 Thin Layer Chromatography (TLC) .......................................................................... 16
3 Results .............................................................................................................................. 17
3.1 Sequences and alignments ......................................................................................... 17
3.2 Phylogenetic analyses ................................................................................................ 17
3.3 Morphological investigations .................................................................................... 21
3.4 TLC ............................................................................................................................ 21
4 Discussion ........................................................................................................................ 23
4.1 Clade B’: Sporacestra and Phyllopsora .................................................................... 24
4.2 Clade C: Bacidia, Bacidiopsora and Phyllopsora ..................................................... 27
4.3 Conclusion and final remarks .................................................................................... 29
4.4 Further studies ........................................................................................................... 30
5 Taxonomy ......................................................................................................................... 32
Sporacestra A. Massal. ........................................................................................................ 32
Sporacestra isidiata Stapnes & Timdal, sp. nov. ............................................................. 33
Sporacestra longispora Stapnes, sp. nov. ........................................................................ 34
IX
Sporacestra pertexta (Nyl.) Stapnes & Timdal, comb. nov. ............................................ 37
Sporacestra straminea Stapnes, sp. nov........................................................................... 39
Appendix: Bacidia De Not. .................................................................................................. 42
Bacidia corallina Stapnes & Timdal, sp. nov. ................................................................. 42
Bacidia kariegae Stapnes, sp. nov ................................................................................... 43
Bacidia microphylla Stapnes, nom. nov ........................................................................... 44
Bacidia soralifera Stapnes & Timdal, nom. nov. ............................................................ 44
Literature .................................................................................................................................. 47
Appendix .................................................................................................................................. 51
Figure S1: PJ phylogeny of sequence alignment 2 and 3. .................................................... 51
Figure S2: PJ phylogeny of sequence alignment 4 .............................................................. 52
1
1 Introduction
The majority of all undescribed fungal species, including lichenized fungi, are expected in
habitats such as tropical forests (Hawksworth & Rossman, 1997; Lawrey & Diederich, 2003;
Sipman & Aptroot, 2001). Of the ca. 2 million geocoded collections and observations of the
Lecanoromycetes (the largest class of lichenized fungi, containing 78% of all accepted
lichenized species [Lücking et al., 2016]) in GBIF, only 3% are from the tropics, and only
0.08% from the Amazon (E. Timdal, pers. comm.). This clearly shows how under-explored
the tropical habitat is, in particular the Amazon, and that it is likely that many species are still
left to be discovered. (Lücking et al., 2016)
Tropical members of the lichen
family Ramalinaceae remain
poorly studied. A set of genera
occurring almost exclusively in
the Tropics and Subtropics are
currently being studied using
molecular methods by Timdal
and co-workers. An
unpublished preliminary
phylogenetic study based on the
mitochondrial small subunit
(mtSSU) revealed several
strongly supported clades
within the Ramalinaceae (Fig.
1: clade A; Bendiksby &
Timdal, unpubl.). One of those,
here referred to as clade A,
consists of a few species
currently included in the three
genera: (1) the tropical genus
Phyllopsora Müll. Arg. (the
type species P. breviuscula
2
(Nyl.) Müll. Arg excluded), (2) the temperate and tropical genus Bacidia De Not. (the type B.
rosella included; for author citations of the species see Table 1), and (3) the tropical genus
Bacidiopsora Kalb (the type B. squamulosula included). One indetermined species assumed
by Bendiksby & Timdal (unpubl.) to be included in a resurrected genus Sporacestra A.
Massal was also included in clade A. This long unused genus name is typified on Biatora
prasina Tuck. & Mont. (nom. illeg.), a species later placed in Bacidia by Coppins (1983). In
Ekman (1996: p. 43) it is stated that: “In fact, the only species included in Sporacestra by
Massalongo, Biatora prasina Tuck. & Mont. (a nomen illegitimum), is synonymous with
Psorella pertexta, a fact which has been overlooked up until now. [….] I have seen the types of
nearly all South and Central American Bacidia s. lat. with long and septate spores, but no
species is close to Psorella pertexta” , where Psorella pertexta (Nyl.) Müll. Arg. is a synonym
of Phyllopsora pertexta (Nyl.) Swinscow & Krog. On the basis of this, it was assumed by
Bendiksby & Timdal (unpubl.) that Sporacestra should be resurrected. Clade A (Fig. 1) is the
topic of this master thesis.
Bacidia is a large genus of crustose species with a world-wide distribution. It consists of
c. 230 species (Lücking et al., 2016), most of them corticolous. The main diagnostic character
for the genus is the morphology of the ascospores: thick-walled and multiseptate. The most
comprehensive study of the genus to date is the PhD thesis on all species of Bacidia and
Bacidina in North America by Ekman (1996). A search in the Recent Literature on Lichens
database (http://nhm2.uio.no/lichens) displays 386 publications on Bacidia, most of which are
taxonomic papers or checklists. In a molecular study based on the nuclear ribosomal internal
transcribed spacer (ITS), monophyly of Bacidia was rejected, with members of Bacidina
Vézda and Toninia A. Massal. nested into Bacidia (Ekman, 2001). There is no study on
Bacidia to date where Bacidiopsora or Phyllopsora are included. At the start of this study,
there were 157 DNA sequence entries of 29 Bacidia species in GenBank.
Bacidiopsora is a tropical/subtropical genus consisting of six rarely collected species
(Lücking et al., 2016). When sterile, Bacidiopsora species are more or less morphologically
indistinguishable from Phyllopsora in the field. When fertile, the apothecia are more flattened
and with a better developed margin. The main distinguishing characters however, are in the
ascospores: thick-walled and multi-septate in Bacidiopsora, and thin-walled and simple in
Phyllopsora. Moreover, Bacidiopsora differs from Bacidia in forming a squamulose thallus
and containing hyperhomosekikaic and homosekikaic acid, whereas Bacidia mostly forms a
3
crustose thallus and contains no lichen substances or rarely atranorin and zeorin (Ekman,
1996; Kalb & Elix, 1995). There are few studies on Bacidiopsora (A. Aptroot, 2002; Brako,
1991; Kalb, 1988, 2004; Kalb & Elix, 1995), most of which are reports on new species,
including discussions about its relationship to other members of the Ramalinaceae based on
morphology, anatomy and chemistry. No molecular studies are published to this date and
therefore no DNA sequence entries of Bacidiopsora exist in GenBank.
Phyllopsora is a genus consisting of crustose to squamulose species, occurring primarily in
Tropical and Subtropical humid woodland and rainforests. More than 120 species names exist
in the literature (Timdal, 2011). The genus has been revised by Swinscow and Krog (1981),
Brako (1989, 1991), Timdal and Krog (2001), and Timdal (2008, 2011), but only with
morphological, anatomical and chemical methods. A preliminary molecular phylogenetic
analysis of a broad sampling of Phyllopsora species and selected presumed relatives
(Bendiksby & Timdal, unpubl.: Fig. 1) indicates that the genus is highly polyphyletic. A
molecular phylogenetic study on the genus as a whole is in preparation by Kistenich et al.
Phyllopsora borbonica and P. pertexta belong in clade A (Fig. 1). The former is
Paleotropical, the latter Neotropical. It has been indicated by anatomical studies (Timdal,
2011) that the two species may in-fact be synonymous. Comparing the species’ descriptions,
the latter differs from the former mainly in the pigmentation in the hypothecium within the
ascocarp (cfr. Swinscow & Krog, 1981; Timdal & Krog, 2001).
Brako (1989) indicated that P. pertexta belonged in an undescribed genus together with P.
cognata (Nyl.) Timdal and P. microphyllina (Nyl.) Swinscow & Krog, due to anatomical
differences separating them from Phyllopsora. Timdal (2008, 2011) applied a broader genus
concept for Phyllopsora, regarding the anatomical differences outlined by Brako (1989) not
being sufficient for splitting the genus. Unfortunately, P. cognata and P. microphyllina are
both unambiguously known only from their type collections (Cuba, localities unknown, leg.
C. Wright, in the 1850s or early 1860s).
A third Phyllopsora species, P. sorediata, also seems to be related to Bacidia and
Bacidiopsora (Fig. 1: clade A). The species was originally described in the genus Triclinum
Fée by Aptroot et al. (2007), which Timdal (2011) regarded as synonymous with the genus
Phyllopsora. The type species of Triclinum, P. cinchonarum (Fée) Timdal, currently studied
by Kistenich and coworkers, seems to belong in a different clade within the Ramalinaceae
4
(S. Kistenich, pers. comm.). Hence, the generic position of P. sorediata is in need of revision,
as it is neither a Phyllopsora nor a Triclinum.
The aim with the present study is to reveal phylogenetic relationships within clade A (Fig. 1)
and to use this phylogenetic hypothesis to assess the taxonomic affiliation and species
delimitations in this clade. With basis in the preliminary molecular phylogeny by Bendiksby
& Timdal (unpubl.), I will expand the dataset, both with regard to taxon sampling and genetic
data, in order to increase resolution and support for phylogenetic relationships. Attaining an
integrative taxonomic approach (Dayrat, 2005), I will investigate the morphology, anatomy,
and chemistry of clade A. Moreover, I will relate these data to the molecular phylogenetic
hypothesis in the work towards potential taxonomic changes. Some obvious hypotheses to be
tested include: (1) Sporacestra represents a monophyletic genus; (2) Phyllopsora borbonica is
synonymous with P. pertexta; (3) Bacidiopsora sp. 1 represents a new genus; (4) Phyllopsora
sorediata represents a new genus; (5) Bacidiopsora is monophyletic; (6) Bacidiopsora is not
nested within Bacidia.
5
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AF
282
084
X2*
X2*
X*
X2*
- X*
- X*
X2
*
- - - - - - KX
098
339
SE
93
SE
23
EU
516
945
SE
306
SE
11
7
SE
50
DQ
98
68
08
DQ
97
29
98
JQ796
832
- X*
XB
*,
X3R
*,
XR
*
- XB
*
X*
XR
*
XR
*
X*
XF
*
X*
XA
*
X*
X*
XB
*
Tab
le 1
: Spec
imen
s use
d i
n t
his
stu
dy
wit
h v
ouch
er i
nfo
rmati
on (
extr
act
ion n
um
be
r, s
pec
ies
nam
es, co
untr
y, l
oca
tion, co
llec
tor,
coll
ecti
on n
um
ber
or
her
bari
a n
um
ber
wit
h h
erbari
a l
oca
tion, ty
pe
info
rmati
on, m
ajo
r li
chen
subst
ance
s
an
d G
eneB
an
k a
cces
sio
n n
um
ber
or
sym
bo
l in
dic
ati
ng
a n
ewly
gen
era
ted
seq
uen
ce o
f ei
ther
mtS
SU
or
ITS
). B
old
fa
ce i
nd
ica
tes
tha
t th
e se
qu
ence
or
chem
istr
y d
ata
are
gen
era
ted
in
th
is s
tud
y.
Abbre
vati
ons:
Ext
r.# =
ext
ract
ion n
um
ber,
Coll
.# =
coll
ecti
on n
um
ber
, H
b.#
= h
erbari
um
num
ber.
X =
seq
uen
ce s
ucc
essf
ull
y g
ener
ate
d,
lett
ers
or
num
ber
s fo
llow
ing X
indic
ate
s th
at
only
part
s o
f th
e D
NA
wer
e su
cces
sfull
y se
quen
ced;
1 =
only
IT
S1, 2 =
only
IT
S2, 4 =
only
IT
S 4
, 5 =
only
IT
S 5
, 3R
= o
nly
mtS
SU
3R
, A
= o
nly
mtS
SU
A, B
= o
nly
mtS
SU
B , F
= o
nly
mtS
SU
-F, L
R =
only
IT
S-l
ichR
,
R =
only
mts
suR
, * i
ndic
ate
s th
at
the
sequen
ce i
s n
ot
incl
uded
in t
his
stu
dy, -
indic
ate
s th
at
ther
e w
ere
no s
ucc
essf
ull
am
pli
fici
ati
on o
f D
NA
.
6
EX
TR
. #
S
PE
CIE
S
CO
UN
TR
Y L
OC
AT
ION
C
OL
LE
CT
OR
C
OL
L.#
or
Hb.#
(HB
)T
YP
E
C
HE
MIS
TR
Y m
tSS
U I
TS
Tab
le 1
: co
nti
nued
.
- - - - - -
5761
6030
6034
6035
6036
6033
6073
6074
6011
6013
6026
6027
6028
6029
6031
6032
6075
6076
6080
48
05
48
06
10
14
10
15
14
28
60
37
60
78
60
79
60
40
60
41
60
23
60
25
6024
508
Baci
dia
sp.2
Baci
dia
su
bin
com
pta
(Nyl.
Arn
old
)1
Baci
dia
su
bin
com
pta
2
Baci
dia
su
ffusa
(F
r.)
Sch
nei
der
Baci
dia
ve
rmif
era
(N
yl.
)T
h. F
r.1
Baci
dia
ve
rmif
era
2
Baci
dia
w
elli
ngto
nii
(Sti
rt.)
D. J.
Gal
low
ay
Baci
dio
pso
ra m
icro
phyl
lina
Kal
b
Baci
dio
pso
ra m
icro
phyl
lina
Baci
dio
pso
ra m
icro
phyl
lina
Baci
dio
pso
ra m
icro
phyl
lina
Baci
dio
pso
ra m
icro
phyl
lina
Baci
dio
pso
ra m
icro
phyl
lina
Baci
dio
pso
ra m
icro
phyl
lina
Baci
dio
pso
ra o
riza
bana
(Vai
n.)
Kal
b
Ba
cid
iop
sora
pso
rin
a(N
yl.
) K
alb
Ba
cid
iop
sora
pso
rin
a
Ba
cid
iop
sora
pso
rin
a
Ba
cid
iop
sora
pso
rin
a
Ba
cid
iop
sora
pso
rin
a
Ba
cid
iop
sora
sil
vico
la (
mal
me)
Kal
b
Ba
cid
iop
sora
sil
vico
la
Ba
cid
iop
sora
sp.
Ba
cid
iop
sora
sp.
Ba
cid
iop
sora
sp.
Ba
cid
iop
sora
sp.
Ba
cid
iop
sora
sp.
Baci
dio
pso
rasp
. 1
Baci
dio
pso
rasp
. 1
Baci
dio
pso
rasp
. 1
Baci
dio
pso
rasp
. 2
Baci
dio
pso
rasp
. 2
Baci
dio
pso
rasp
. 2
Baci
dio
pso
rasp
. 3
Baci
dio
pso
rasp
. 3
Baci
dio
pso
ra s
quam
ulo
sula
(Nyl.
) K
alb.
Baci
dio
pso
ra s
quam
ulo
sula
Baci
dio
pso
ra s
quam
ulo
sula
Baci
dio
pso
ra s
quam
ulo
sula
Sw
iter
land
Sw
iter
land
Sw
eden
- - Un
ited
Kin
gd
om
New
Zea
lan
d
Co
sta
Ric
a
Ven
ezu
ela
Cam
ero
on
Réu
nio
n
Bra
zil
So
uth
Afr
ica
So
uth
Afr
ica
Th
aila
nd
Sri
Lan
ka
Ven
ezuel
a
Ecu
ador
Guat
emal
a
Bra
zil
Bra
zil
Ecu
ador
Ven
ezuel
a
Ven
ezuel
a
South
Afr
ica
South
Afr
ica
South
Afr
ica
Thai
land
Thai
land
Thai
land
South
Afr
ica
South
Afr
ica
South
Afr
ica
Ven
ezuel
a
Ven
ezuel
a
Cost
a R
ica
Ecu
ado
r
Bra
zil
Ecu
ado
r
Wai
an R
iver
, M
anap
ouri
Punta
renas
Tac
hir
a
Nort
hW
est
pro
vin
ce, M
ount
Oku
- Min
as G
erai
s
Eas
tern
Cap
e,W
este
rn D
istr
ict,
11 k
m N
NW
of
Ken
ton-o
n-S
ea
Eas
tern
Cap
e,W
este
rn D
istr
ict,
11 k
m N
NW
of
Ken
ton-o
n-S
ea
Do
i C
hia
ng
Dao
- Mer
ida
Azu
ay
Ch
imal
ten
ang
o
Min
as G
erai
s
São
Pau
lo
Azu
ay
Mir
and
a, C
arac
as,
El
Vo
lcan
Mir
and
a, C
arac
as,
El
Vo
lcan
Mp
um
alan
ga,
Eh
lan
zen
i D
istr
ict,
Bu
ffel
klo
of
Nat
ure
Res
erv
e
Mp
um
alan
ga,
Eh
lan
zen
i D
istr
ict,
gra
ssla
nd
ab
ov
e S
ud
wal
a C
aves
Mp
um
alan
ga,
Eh
lan
zen
i D
istr
ict,
Bu
ffel
klo
of
Nat
ure
Res
erv
e
Uth
aiT
han
i, K
hlo
ng
Plo
u
Uth
aiT
han
i, K
hao
Nan
g R
um
Uth
aiT
han
i, K
hlo
ng
Plo
u
Mpum
alan
ga,
Ehla
nze
ni
Dis
tric
t, B
uff
elklo
of
Nat
ure
Res
erve
Mpum
alan
ga,
Ehla
nze
ni
Dis
tric
t, B
uff
elklo
of
Nat
ure
Res
erve
Mpum
alan
ga,
Ehla
nze
ni
Dis
tric
t, B
uff
elklo
of
Nat
ure
Res
erve
Cap
ital
Dis
tric
t, P
arque
Nac
ional
War
aira
Rep
ano
Cap
ital
Dis
tric
t, P
arque
Nac
ional
War
aira
Rep
ano
San
José
Azu
ay
Min
as G
erai
s
Azu
ay
- - Ekm
an, S
.
Lu
mb
sch
, H
., -
- CJ
Ell
is &
BJ
Co
pp
ins
Ziv
iag
ina,
N.
Kal
b,
K.
& P
löb
st, G
.
Kal
b,
K.
& K
alb
,A
.
Fri
sch
,A.
&T
amn
jon
g I
di
Kal
b,
K.
& K
alb
,A
.
Kal
b,
K.
& P
löb
st, G
.
Ru
i, S
. &
Tim
dal
, E
.
Ru
i, S
. &
Tim
dal
, E
.
All
en,
D.
Aptr
oot,
A.
Kal
b, K
., K
alb,A
. &
López
-Fig
uei
ras
Kal
b, K
. &
Kal
b,A
.
Kal
b, K
. &
Plö
bst
, G
.
Kal
b, K
. &
Plö
bst
, G
.
Kal
b, K
. &
Plö
bst
, G
.
Kal
b, K
. &
Kal
b,A
.
Dah
l, K
iste
nic
h,
Tim
dal
&T
ore
skaa
s
Dah
l, K
iste
nic
h,
Tim
dal
&T
ore
skaa
s
Burr
ow
s, J
. &
Tim
dal
, E
.
Burr
ow
s, J
. &
Tim
dal
, E
.
Burr
ow
s, J
. &
Tim
dal
, E
.
Aguir
re, Ja
mes
&W
ols
eley
Aguir
re, Ja
mes
&W
ols
eley
Aguir
re, Ja
mes
&W
ols
eley
Burr
ow
s, J
. &
Tim
dal
, E
.
Burr
ow
s, J
. &
Tim
dal
, E
.
Burr
ow
s, J
. &
Tim
dal
, E
.
Dah
l, K
iste
nic
h,
Tim
dal
&T
ore
skaa
s
Dah
l, K
iste
nic
h,
Tim
dal
&T
ore
skaa
s
Kal
b,
K.
& P
löb
st, G
.
Kal
b,
K.
& K
alb
,A
.
Kal
b,
K.
& P
löb
st, G
.
Kal
b,
K.
& K
alb
,A
.
- - 3413 (
BG
)
19
19
c, -
- L6
29
: 1
3 (
-)
L-2
04
59
8 (
O)
12
21
88
(W
IS)
12
21
97
(W
IS)
12
21
95
(W
IS)
12
21
96
(W
IS)
12
21
86
(W
IS)
L-2
02
86
8 (
O)
L-2
02
86
7 (
O)
76
49
74
(B
M)
733846 (
BM
)
122194 (
WIS
)
122192 (
WIS
)
122193 (
WIS
)
122190 (
WIS
)
122187 (
WIS
)
122189 (
WIS
)
L-2
02459 (
O)
L-2
02458 (
O)
L-1
96765 (
O)
L-1
96740 (
O)
L-1
96810 (
O)
2478a
(BM
)
749853 (
BM
)
1031544 (
BM
)
L-1
96747 (
O)
L-1
96757 (
O)
L-1
96762 (
O)
L-2
02485 (
O)
L-2
02
48
4 (
O)
12
22
00
(W
IS)
12
22
01
(W
IS)
12
21
99
(W
IS)
L-1
13
54
3 (
O)
Ty
pe
spec
ies
of
Ba
cid
iop
sora
HS
EK
HH
SE
K
HH
SE
K, H
SE
K
HS
EK
HH
SE
K,
HS
EK
HH
SE
K/H
SE
K
HH
SE
K/H
SE
K
NO
NE
HH
SE
K,
HS
EK
, S
EK
HH
SE
K,
HS
EK
, S
EK
HH
SE
K,
HS
EK
HH
SE
K,
HS
EK
HS
EK
HH
SE
K,
HS
EK
UN
KN
NO
NE
HH
SE
K/H
SE
K
HH
SE
K
HH
SE
K/H
SE
K
HH
SE
K/H
SE
K, X
AN
TH
HH
SE
K/H
SE
K, X
AN
TH
HH
SE
K/H
SE
K, X
AN
TH
HH
SE
K/H
SE
K
HH
SE
K/H
SE
K
HH
SE
K/H
SE
K
HH
SE
K/H
SE
K
HH
SE
K/H
SE
K
HH
SE
K, H
SE
K, S
EK
HH
SE
K, H
SE
K, S
EK
HH
SE
K, H
SE
K, S
EK
HH
SE
K/H
SE
K
KJ7
66
35
5
X - - - - - XB
XB
XB
*
- - XB
*
- - - - X*
X*
X - XB
*
X X X X XB
XB
X X - XB
*
- X
KX
09
83
40
KX
09
83
42
AF
28
21
25
AF
28
20
91
AF
28
21
09
FR
79
91
29
- - - - - X1
X1
- - - X2*
X2*
- - - X*
- - - - X - - - X X - - - X
Ty
pe
spec
ies
of
Pso
rell
a
7
EX
TR
. #
SP
EC
IES
C
OU
NT
RY
LO
CA
TIO
N C
OL
LE
CT
OR
C
OL
L.#
or
Hb.#
(HB
)T
YP
E
C
HE
MIS
TR
Y m
tSS
U IT
S
Tab
le 1
: co
nti
nu
ed.
5636
6017
5483
511
6012
427
6021
1040
5638
5641
6015
6016
6043
6044
6085
6087
6086
5622
5623
5624
1433
5637
1008
1007
6009
6010
6014
6019
6020
6022
1312
395
6084
5613
5615
5616
5620
5625
5639
5640
6018
Baci
dio
pso
ra t
enuis
ecta
(Vai
n.)
Aptr
oot
Baci
dio
spora
ori
zabana
Luek
ingia
poly
spora
Aptr
oo
t &
Um
ana
Phyl
lopso
ra b
orb
onic
aT
imdal
& K
rog
Phyl
lopso
ra b
orb
onic
a
Phyl
lopso
ra b
orb
onic
a
Ph
yllo
pso
ra c
on
fusa
Sw
insc
ow
& K
rog
Ph
yllo
pso
ra p
erte
xta
(N
yl.
) S
win
sco
w &
Kro
g
Ph
yllo
pso
ra p
erte
xta
Ph
yllo
pso
ra p
erte
xta
Ph
yllo
pso
ra p
erte
xta
Ph
yllo
pso
ra p
erte
xta
Ph
yllo
pso
ra p
erte
xta
Ph
yllo
pso
ra p
erte
xta
Phyl
lopso
ra p
erte
xta
Phyl
lopso
ra p
erte
xta
Phyl
lopso
ra p
erte
xta
Phyl
lopso
ra s
ora
life
raT
imdal
Phyl
lopso
ra s
ora
life
ra
Phyl
lopso
ra s
ora
life
ra
Phyl
lopso
ra s
ore
dia
ta(A
ptr
oot
& S
par
rius)
Tim
dal
Ph
yllo
pso
ra s
ore
dia
ta
Ph
yllo
pso
ra s
ore
dia
ta
Ph
yllo
pso
ra s
ore
dia
ta
Ph
yllo
pso
ra s
p.
Ph
yllo
pso
ra s
p.
Ph
yllo
pso
ra s
p.
Ph
yllo
pso
ra s
p.
Phyl
lopso
ra s
p.
Phyl
lopso
ra s
p.
Phys
cidia
cyl
ind
rophora
Phys
cidia
sp.
Phys
cidia
sp.
Ram
alin
acea
e in
det
.
Ram
alin
acea
e in
det
.
Ram
alin
acea
e in
det
.
Ram
alin
acea
e in
det
.
Ram
alin
acea
e in
det
.
Ram
alin
acea
e in
det
.
Ram
alin
acea
e in
det
.
Ram
alin
acea
e in
det
.
Bra
zil
Th
aila
nd
Co
sta
Ric
a
La
Réu
nio
n
Sey
chel
les
Indones
ia
Cam
eroon
Cuba
Pap
ua
New
Guin
ea
Pap
ua
New
Guin
ea
Pap
ua
New
Gu
inea
Pap
ua
New
Gu
inea
Ven
ezu
ela
Ven
ezu
ela
Bra
zil
Bra
zil
Bra
zil
Bra
zil
Bra
zil
Bra
zil
South
Afr
ica
Thai
land
Thai
land
Thai
land
Mal
aysi
a
Mal
aysi
a
Bra
zil
Cam
ero
on
Tan
zan
ia
Bra
zil
Tai
wan
Cu
ba
Mal
aysi
a
Bra
zil
Bra
zil
Bra
zil
Bra
zil
Bra
zil
Cam
eroon
Cam
ero
on
Cam
ero
on
Cat
asA
ltas
, P
arq
ue
Nat
ura
l d
o C
arac
a
Uth
aiT
han
i, K
hao
Nan
g R
um
Lim
ón
, H
ito
y C
erer
e R
eser
ve,
nea
r P
and
rora
,
alo
ng r
oad
to
war
ds
Pla
ine
d'A
ffo
uch
es,
abo
ve
Bra
s C
itro
n
Mah
é, M
are
aux c
och
ons
Kal
iman
tan S
elan
, H
ulu
Tab
along, si
te 4
Nort
hW
est
pro
vin
ce, D
onga-
Man
tunga
Div
isio
n
Pin
ar d
el R
ío,L
a P
alm
a, M
ogote
el
Pan
de
Guaj
aibón
Moro
be,
Her
zog M
ts,
Wag
o
Moro
be,
Bulu
lo
Mo
rob
e, B
ewap
i
Mo
rob
e, M
t. S
elk
Cap
ital
Dis
tric
t, P
arq
ue
Nac
ion
alW
arai
ra R
epan
o
Cap
ital
Dis
tric
t, P
arq
ue
Nac
ion
al M
acar
ao
Rio
Ab
ajo
Rio
Ab
ajo
Rio
Ab
ajo
Par
á, P
arag
om
inas
, H
yd
ro m
inin
g a
rea
Par
á, P
arag
om
inas
, H
yd
ro m
inin
g a
rea
Par
á, M
elgaç
o, F
lore
sta
Nac
ional
de
Cax
iuan
ã,
Eas
tern
Cap
e, G
rootr
ivie
r
Uth
aiT
han
i, K
hlo
ng P
lou
Uth
aiT
han
i, K
hlo
ng P
lou
Uth
aiT
han
i, K
hao
Nan
g R
um
Born
eo, S
abah
, M
alia
u B
asin
Bo
rneo
, S
abah
, S
AF
E-p
roje
ctA
rea
Rio
Ab
ajo
Eas
t p
rov
ince
,Y
ok
adu
ma
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rog
oro
Reg
ion
Rio
Ab
ajo
Tai
chu
ng
co
. M
t.Y
uan
azu
ei
Ho
lgu
ín,
Moa,
Par
qu
e N
acio
nal
Ale
jan
dro
de
Hu
mb
old
t
Bo
rneo
, S
abah
, M
alia
u B
asin
Par
á, P
arag
om
inas
, H
ydro
min
ing a
rea
Par
á, M
elgaç
o, F
lore
sta
Nac
ional
de
Cax
iuan
ã
Par
á, M
elgaç
o, F
lore
sta
Nac
ional
de
Cax
iuan
ã
Par
á, M
elgaç
o, F
lore
sta
Nac
ional
de
Cax
iuan
ã
Par
á, M
elgaç
o, F
lore
sta
Nac
ional
de
Cax
iuan
ã
South
wes
t pro
vic
e, F
ako D
ivis
ion
Lit
tora
l P
rov
ince
, D
ou
ala-
Ed
ea F
ore
st R
eser
ve
No
rth
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t p
rov
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isio
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Ap
tro
ot,
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Ag
uir
re,
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es &
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lsel
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ver,
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sch
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g I
di
Pér
ez-O
rteg
a, S
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my,
A.C
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l, D
. J.
Jere
my,
A.C
.
Hil
l, D
. J.
Dah
l, K
iste
nic
h,
Tim
dal
&T
ore
skaa
s
Dah
l, K
iste
nic
h,
Tim
dal
&T
ore
skaa
s
Các
eres
, M
.
Các
eres
, M
.
Các
eres
, M
.
Bar
bo
sa,
R.S
., H
aug
an,
R.
&T
imd
al,
E.
Bar
bo
sa,
R.S
., H
aug
an,
R.
&T
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al,
E.
Kis
ten
ich
, S
. &
Tim
dal
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No
rdin
,A
.
Ag
uir
re,
Jam
es &
Wo
lsel
ey
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uir
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es &
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lsel
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Aguir
re &
Wols
eley
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eley
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Các
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mu
ra,Y
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ck,W
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rap
pan
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bo
sa,
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., H
aug
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al,
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Kis
ten
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, S
. &
Tim
dal
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ten
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Tim
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Kis
ten
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Tim
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ten
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dal
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sch,A
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njo
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di
Fri
sch,A
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njo
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di
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sch,A
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Tam
njo
ng I
di
5030069994830 (
BR
)
763699 (
BM
)
5030070745698 (
BR
)
L-7
97 (
O)
11
43
07
(h
b S
eaw
ard
)
T1
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Q (
BM
)
99
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(h
bA
. F
risc
h)
s.n
. (h
b P
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ega)
577345 (
BM
)
921669 (
BM
)
577330 (
BM
)
921683 (
BM
)
L-2
02494 (
O)
L-2
02519 (
O)
20131 (
ISE
)
20134 (
ISE
)
20
110 (
ISE
)
L-1
939
72 (
O)
L-1
939
24 (
O)
L-2
010
89 (
O)
L-0
926
04 (
UP
S)
763640 (
BM
)
749391 (
BM
)
749765 (
BM
)
M0
87 (
BO
RH
)
S.E
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OR
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20109 (
ISE
)
99/K
a2774 (h
bA
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99/T
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bA
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20107 (
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7091 (
GZ
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11
49
47
7 (
NY
)
L5
04 (
BM
)
L-1
938
72 (
O)
L-2
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39 (
O)
L-2
010
91 (
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L-2
010
92 (
O)
L-2
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21 (
O)
99
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99/K
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A. F
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h)
IT o
fL
. p
oly
spo
ra
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of
P.
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HH
SE
K/H
SE
K
NO
NE
NO
NE
NO
NE
TE
RP
NO
NE
NO
NE
NO
NE
NO
NE
NO
NE
FU
R
AT
R
NO
NE
NO
NE
NO
NE
NO
NE
NO
NE
NO
NE
DIV
,AR
G
DIV
,AR
G
DIV
,AR
G
DIV
,AR
G
NO
NE
NO
NE
TE
RP
NO
NE
NO
NE
TE
RP
AT
R,
ZE
O
TE
RP
NO
NE
NO
NE
AR
G
NO
NE
UN
KN
NO
NE
NO
NE
- - X X - XA
XB
*
X - XB
*
- - XB
*
XB
*
XB
*
XB
XB
*
- X*
X*
X XB
*
X X - XB
*
XB
*
- XB
*
X*
X*
X XB
*
XB
*
X3
*
- XB
*
XA
*
- - -
- - X - X2
- X - - - - - X*
- X - - X2*
X1*, X
4*
- - - - - - X5
*
X*
- X2*
X*
- X2*
X2*
- - -
Ab
bri
vat
ion
s: I
T =
Iso
typ
e, H
T =
Ho
loty
pe
8
EX
TR
. #
S
PE
CIE
S
CO
UN
TR
Y L
OC
AT
ION
C
OL
LE
CT
OR
C
OL
L.#
or
Hb.#
(HB
)T
YP
E C
HE
MIS
TR
Y m
tSS
U IT
S
Tab
le 1
: co
nti
nued
.
60
42
60
77
60
88
12
85
56
12
56
14
56
17
56
18
56
19
56
26
56
31
56
32
56
35
60
38
60
39
60
46
60
81
60
82
60
83
56
21
56
28
56
34
-
Ram
alin
acea
e
Ram
alin
acea
e
Ram
alin
acea
e
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Spora
cest
ra s
p.
Tonin
ia c
andid
a (
Web
er)
Th. F
r.
Ven
ezuel
a
Mal
aysi
a
Mal
aysi
a
Mal
aysi
a
Bra
zil
Bra
zil
Bra
zil
Bra
zil
Bra
zil
Bra
zil
Bra
zil
Bra
zil
Bra
zil
Mal
aysi
a
Mal
aysi
a
Bra
zil
Mal
aysi
a
Mal
aysi
a
Mal
aysi
a
Bra
zil
Bra
zil
Bra
zil
Norw
ay
Mir
anda,
Car
acas
, E
lV
olc
an
Born
eo, S
abah
, M
alia
u B
asin
Born
eo, S
abah
, D
anum
val
ley
Born
eo, S
a b
ah, M
alia
u B
asin
Par
á, M
elgaç
o, F
lore
sta
Nac
ional
de
Cax
iuan
ã
Par
á, M
elgaç
o, F
lore
sta
Nac
ional
de
Cax
iuan
ã
Par
á, M
elgaç
o, F
lore
sta
Nac
ional
de
Cax
iuan
ã
Par
á, M
elgaç
o, F
lore
sta
Nac
ional
de
Cax
iuan
ã
Par
á, M
elgaç
o, F
lore
sta
Nac
ional
de
Cax
iuan
ã
Par
á, M
elgaç
o, F
lore
sta
Nac
ional
de
Cax
iuan
ã
Par
á,T
hai
lândia
, F
azen
da
Agro
ecoló
gic
a S
ão R
oque
Par
á,T
hai
lândia
, F
azen
da
Agro
ecoló
gic
a S
ão R
oque
Par
á,T
hai
lândia
, F
azen
da
Agro
ecoló
gic
a S
ão R
oque
Born
eo, S
abah
, K
inab
alu p
ark
Born
eo, S
abah
, K
inab
alu p
ark
Par
á,T
hai
lândia
, F
azen
da
Agro
ecoló
gic
a S
ão R
oque
Born
eo, S
abah
, M
alia
u B
asin
Born
eo, S
abah
, M
alia
u B
asin
Born
eo, S
abah
, M
alia
u B
asin
Par
á, M
elgaç
o, F
lore
sta
Nac
ional
de
Cax
iuan
ã
São
Pau
lo, P
arque
Est
adual
das
Fonte
s do I
pir
anga
Par
á,T
hai
lândia
, F
azen
da
Agro
ecoló
gic
a S
ão R
oque
Dah
l, K
iste
nic
h,
Tim
dal
&T
ore
skaa
s
Vai
rappan
, C
.
Thüs,
Wols
eley
&V
aira
ppan
Thüs,
Wols
eley
&V
aira
ppan
Kis
tenic
h, S
. &
Tim
dal
, E
.
Kis
tenic
h, S
. &
Tim
dal
, E
.
Kis
tenic
h, S
. &
Tim
dal
, E
.
Kis
tenic
h, S
. &
Tim
dal
, E
.
Kis
tenic
h, S
. &
Tim
dal
, E
.
Kis
tenic
h, S
. &
Tim
dal
, E
.
Dah
l, K
iste
nic
h,
Tim
dal
&T
ore
skaa
s
Dah
l, K
iste
nic
h,
Tim
dal
&T
ore
skaa
s
Dah
l, K
iste
nic
h,
Tim
dal
&T
ore
skaa
s
Pau
kov,
A.
Pau
kov,
A.
Dah
l, K
iste
nic
h,
Tim
dal
&T
ore
skaa
s
Thüs,
Wols
eley
&V
aira
ppan
Thüs,
Wols
eley
&V
aira
ppan
Thüs,
Wols
eley
&V
aira
ppan
Kis
tenic
h, S
. &
Tim
dal
, E
.
Dah
l, K
iste
nic
h,
Tim
dal
&T
ore
skaa
s
Dah
l, K
iste
nic
h,
Tim
dal
&T
ore
skaa
s
Bra
tli
&T
imdal
L-2
02457 (
O)
L257 (
BM
)
D.8
.07.1
(B
OR
H)
M.3
.08.o
Q.2
(B
OR
H)
L-2
01033 (
O)
L-2
01106 (
O)
L-2
01020 (
O)
L-2
01048 (
O)
L-2
01101 (
O)
L-2
01109 (
O)
L-2
02588 (
O)
L-2
02596 (
O)
L-2
02591 (
O)
2231 (
hb P
aukov)
2227 (
hb P
aukov)
L-2
02588 (
O)
M.3
.01.2
(B
OR
H)
M.3
.03.2
(B
OR
H)
M132 (
BO
RH
)
L-2
04486 (
O)
L-2
02768 (
O)
L-2
02576 (
O)
L-2
5779 (
O)
NO
NE
- NO
NE
NO
NE
NO
NE
NO
NE
NO
NE
NO
NE
NO
NE
NO
NE
FA
T
NO
NE
NO
NE
NO
NE
NO
NE
FA
T
NO
NE
NO
NE
NO
NE
UN
KN
UN
KN
+T
ER
P
NO
NE
-
X*
- - X X X XA
, X
R
X X XA
*
- XA
- X*
XB
*
- - - XB
*
- X*
X*
X1*
- - X2
X X2
X X X2*
- X X*
- - - X*
- - X2*
X5*, X
LR
*
X1*
SE
59
9
Table 2. An overview of field work locations in Venezuela and Brazil.
Country Location Habitat Altitude
Venezuela Miranda, Caracas, El Volcán Carribean moist forest 1430-1480 m alt.
Capital District, Parque Nacional Waraira Repano Carribean moist forest 1800-2040 m alt.
Capital District, Parque Nacional Macarao Carribean moist forest 1860-2080 m alt.
Carabobo, Parque Nacional Henry Pittier Carribean rainforest 1150-1270 m alt.
Brazil Parà, Tailândia, Fazenda Agroecológica Sâo Roque Amazonian rainforest 50-60 m alt.
Rio de Janeiro, Parque Nacional do Itatiaia Atlantic rainforest 800-1200 m alt.
São Paulo, Parque Estadual das Fontes do Ipiranga Atlantic rainforest 790-830 m alt.
São Paulo, Reserva Biológica do Alto da Serra de Paranapiacaba Atlantic rainforest 790-810 m alt.
2 Material and methods
2.1 Fieldwork
The fieldwork was conducted in tropical rainforests and tropical moist forests in Venezuela
and Brazil during a period of 25 days in November – December 2015. I travelled with Einar
Timdal, Sonja Kistenich, and Anne Karin Toreskaas, and we were accompanied by local
researchers at all localities. The localities were chosen by the local researchers. An overview
of the eight locations is presented in Table 2.
During fieldwork, the samples were preserved to prevent deterioration. We packed the
collected specimens in acid free paper capsules and placed these between weighted wool
cardboard in order to desiccate and flattened the specimens. After drying, the specimens were
placed in sealed plastic bags with silica gel (as recommended by Chase & Hills, 1991).
The collections were made under local research and collection permits. The specimens
collected in Venezuela were split in two at Universidad Central de Venezuela (VEN); the first
set was retained in VEN and the second sent to the Natural History Museum in Oslo (O). The
Brazilian collections were sent undivided to O, but will be split after completion of this study.
The first set will be returned to Museu Paraense Emílio Goeldi (MG; the specimens from
Pará) and Universidad de Federal de Sergipe (ISE; the specimens from São Paulo and Rio de
Janeiro); the second set will be stored in O.
10
2.2 The specimens
The study is based on both newly collected specimens from Venezuela and Brazil and
archived specimens from various herbaria. The fieldwork provided in total 29 newly collected
specimens that were thought to belong in clade A, namely Sporacestra, Bacidia and
Bacidiopsora. Due to the great morphological similarity of the genera, identification in field
proved to be challenging. Ninety-six herbarium specimens suspected to belong in the same
focus taxa (age up to 50 years) held at the following herbaria were also included: BM, BORH,
BR, ISE, O, UPS, WIS, and the private herbaria of A. Frisch, A. Paukov, S. Pérez-Ortega and
M. Seaward (herbarium abbreviations follow Holmgren et al. 1990). Eighteen sequences were
borrowed from Kistenich et al. (in prep.; ongoing molecular phylogenetic studies of the
Ramalinaceae). Twenty-six sequences of Bacidia were downloaded from GenBank, based on
the taxon sampling in Ekman (2001). Available voucher data for all specimens are provided
in Table 1.
2.3 DNA work
2.3.1 DNA-extraction
Up to 5 mg of tissue was sampled from 96 specimens, both freshly collected and herbarium
specimens. If apothecia were present, they were sampled to ensure DNA originating from the
mycobiont. The samples were placed into 2 mL microcentrifuge tubes with two sterilized
3 mm Tungsten-Carbide Beads (Applied Biosystems™, Foster City, California, U.S.A.),
crushed using a Mixer Mill (MM301, Retsch GmbH & Co., Haan, Germany) and grinded for
2×1 min at 20Hz to ensure pulverized samples. I extracted DNA using the E.Z.N.A.® SP
Plant DNA Kit (Omega Bio-Tek, Georgia, U.S.A) following the manufacturer’s protocol,
including the suggested additional elution step: samples were eluted twice (with 25 µL elution
buffer in each) in two separate Safe-Lock Tubes™ (Eppendorf, Hamburg, Germany). Prior to
final spinning, samples were incubated at 65 ºC for 5 min to increase DNA yield and
concentration. The second eluates serve as back-up and are stored in the DNA collection at
the Natural History Museum, University of Oslo.
11
2.3.2 Polymerase Chain Reaction (PCR)
I performed PCR amplification on all extracted DNAs to amplify the nuclear ribosomal
internal transcribed spacer region (ITS: ITS1, 5.8S and ITS2) and the mitochondrial small
subunit (mtSSU). Thus, including two markers from different genomes as proposed by
Rubinoff and Holland (2005) . These DNA regions are commonly used in lichen systematics
in general (e.g. Crespo & Lumbsch, 2010; Ekman, 2001; Lendemer et al., 2016; Schoch et al.,
2012). Moreover, successful preliminary PCR amplification and sequencing of Sporacestra
and relatives using internal primers for both the ITS (Bendiksby & Timdal, 2013) and mtSSU
(Bendiksby, unpubl.) contributed to my selection of loci.
The ITS region was amplified in one single fragment using the primers ITS4/ITS5 (White et
al., 1990). In case of no visible PCR product, the ITS1 and ITS2 regions were amplified in
two separate reactions using the internal primers ITS-lichR and ITS-lichF (Bendiksby &
Timdal, 2013; Table 3: ITS5 with ITS-lichR and ITS-lichF with ITS4). The first ca. 1000 base
pairs (bp) of the mtSSU was amplified using the primers, mrSSU1 and mrSSU3R (Zoller et
al., 1999). In case of no visible amplification, we amplified the region in two fragments
(mtSSUA and mtSSUB, respectively) using the internal primers mtSSU-R and mtSSU-F with
(Table 3; mtSSU1 with mtSSU-R, and mtSSU-F with mtSSU3R).
12
The amplification of DNA was executede in 12 µL reactions using 0.1 µL AmpliTaq® DNA
polymerase (Applied Biosystems™), 1.25 µL 10x buffer II, purified H2O using Milli-Q®
Integral Water Purification System (Millipore SAS, Molseim, France), 1.25 µL magnesium
chloride (15 mM, MgCl2, Applied Biosystems™), 0.25 µL dinucleotide triphosphate (10 mM,
dNTP, Applied Biosystems™), 0.6 µL bovine serum albumen (1 g/L, BSA), 10 µM of each
primer and 1.5 µL of the extracted DNA.
For some samples, Illustra™ puReTaq Ready-To-Go™ PCR Beads (GE Healthcare,
Buckinghamshire, UK) were used following manufacturers protocol, except for diluting the
content to double the amount of reactions.
The PCR amplifications were run on a 3×T100 Thermal Cycler (Bio-Rad Laboratories, Inc.,
California, U.S.A.) or a GeneAmp® PCR System 9700 (Applied Biosystems™) on the
following cycle conditions: 95 ºC for 10 min, 34 cycles of 95 ºC for 30 s (mtSSU) or 45 s
(ITS), 60 ºC for 30 s, 72 ºC of 30 s, followed by 72 ºC for 7 min and 4 ºC on infinite hold. All
DNA isolates are deposited in the DNA collection at the Natural History Museum, University
of Oslo.
2.3.3 Gel electrophoresis
Amplified PCR products were visualized with electrophoresis on 1% agarose gels, mixing
SeaKem® LE Agarose (Lonza group, Basel, Switzerland) with 0.5×Tris-borate-EDTA-buffer.
GelRed™ nucleic acid dye (Biotum, Hayward, California, U.S.A) was added to stain DNA.
Each well was loaded with 4 µL PCR-product mixed with 4 µL homemade loadingbuffer
(50 mM EDTA, 30% glycerol, 0.25% bromphenol blue and 0.2% xylene cyanol). One well
per row was loaded with 3 µL Fastruler™ Low Range DNA Ladder (Fermentas® Thermo
Fisher Scientific) for sequence length reference.
2.3.4 Cleaning PCR-products
PCR-products were cleaned using a 1:1 proportion of 2 µL of 10× diluted Exonuclease 1
(EXO I, New England Biolabs® inc, Massachusetts, U.S.A) and Shrimp Alkaline
Phosphatase (rSAP, New England Biolabs® inc) to 6 µL PCR product. The mixture was
incubated in the 3×T100 Thermal Cycler (Bio-Rad Laboratories, Inc.) at 37 ºC for 45 min,
then at 80 ºC for 15 min.
13
2.3.5 Cycle sequencing
Cycle sequencing was done using a BigDye Terminator v3.1 Cycle sequencing kit (Applied
Biosystems™). The following master mix for very weak bands were used: 0.4 µL BigDye™
Terminator v3.1 Ready reaction mix (Applied Biosystems™), 1.8 µL 5× Sequencing Buffer
(Applied Biosystems™), 6.3 µL MilliQ water, and 0.5 µL primer (10 µM). The mastermix
was combined with 3.5 µL of cleaned PCR-product, giving a total of 10 µL. The cycle
sequencing reaction was run on a 3×T100 Thermal Cycler (Bio-Rad Laboratories, Inc.) at
96 ºC for 1 min, 30 cycles of 96 ºC for 10 s, 50 ºC for 5 s, 60 ºC for 4 s, and then 4 ºC on
infinite hold.
2.3.6 Ethanol precipitation
Each sequencing product was purified using 2 µL 1:1 mix of 0.125 M EDTA and 3M sodium
acetate (NaAc), and 25 µL of 96% ethanol (EtOH). The samples were vortexed and
centrifuged for 30 min at 4500 revolutions per minute (rpm) at 4 ºC with a plate centrifuge
(Hettich Rotanta 46 RS Centrifuge, Andreas Hettich GmbH & Co. KG). The supernatant was
removed by centrifuging the samples upside down for 15 s at 400 rpm. Then 35 µL of cold
70% EtOH was added and the samples centrifuged at 4500 for 25 s at 4 ºC. Another removal
of the supernatant was executed by repeating the upside down centrifuge step (15 s at
400 rpm). The remaining liquid left in the tubes were removed by using the DNA120
SpeedVac vacuum centrifuge (Savant DNA SpeedVac,Thermo Fisher Scientific,
Massachusets, U.S.A), for 2 min on medium heat. The ethanol precipitated PCR products
were stored at – 20 ºC.
2.3.7 DNA sequencing
Sequencing products were run on an ABI 3130×/ Genetic Analyzer (Applied Biosystems™).
Eleven µL highly deionized formamide (Hi-Di) was added to the ethanol-precipitated
products and left to rest for 15 minutes. Some of the sequencing was outsourced to Macrogen
(Macrogen Europe, Amsterdam, The Netherlands). We used an Illustra™ ExoStar™ (GE
Healthcare) 1-Step kit containing Illustra Alakaline phosphatase and Exonuclease 1, for
cleaning the sequencing products, as recommended by Macrogen.
14
2.3.8 Sequence editing and alignment assembly
Sequences were assembled, thoroughly inspected by eye, and edited manually with Geneious
10.1 (Kearse et al., 2012). The datasets were preliminary aligned using the MAFFT online
interface version 7 (Katoh et al., 2002; Katoh & Standley, 2013), with the multipair strategy.
The default parameter settings were used, except for strategy which was set to –multipair
(Accurate), and the scoring matrix for nucleotide sequences that was set to 20Pam / k = 2.
Extensive manual adjustments were needed and undertaken in BioEdit (Hall, 1999). All
sequences that possessed numerous indels and nucleotides in conflict with the rest of the
dataset, indicating a distant relationship to the focus clade, were excluded.
2.3.9 Data assembly
Four different sequence alignments were assembled and analyzed: (1) A preliminary mtSSU
alignment with 208 accessions that included a broad sampling of the Ramalinaceae (Kistenich
et al., in prep, not shown in voucher table), all newly sequenced specimens, and Bacidia
sequences from GenBank. The main goal of alignment 1 was to examine the phylogenetic
positions and relationships of Sporacestra, Bacidia and Bacidiopsora to other members of the
family. Sequence alignment 1 was important to guide in the species identification as well as in
the selection of which new specimens to include in the study. Based on the preliminary
Ramalinaceae phylogeny (Kistenich et al., in prep), I selected Luckingia polyspora for rooting
in the phylogenetic analyses. (2) An mtSSU subset alignment that was formed by the analysis
of alignment 1. It was assembled to examine the relationship within clade , containing 34
accessions that comprised the newly sequenced specimens, selected sequences borrowed from
Kistenich et al. (in prep.), and Bacidia sequences from GenBank. Luckingia polyspora was
used for rooting. (3) An ITS alignment with 42 accessions where mtSSU alignment 2 guided
the assemblage. It comprised the newly sequenced specimens included in alignment 2,
selected sequences that only successfully amplified the ITS region and belonging to the focus
clade, sequences from Kistenich et al. (in prep.), and Bacidia sequences from GenBank.
Toninia candida was used for rooting based on the sister relationship in the preliminary study
of the Ramalinaceae (Kistenich et al., in prep). (4) A concatenation of alignment 2 and 3,
containing 57 accessions.
15
2.4 Phylogenetic analyses and model selection
I analyzed the data using parsimony jackknifing (PJ) and Bayesian inference (BI)
phylogenetic methods. I perform the PJ-analysis with 1000 replicates using WinClada (Nixon,
2002) through NONA (Goloboff, 1993). The sequence alignments were first run through
SeqState (Müller, 2005) to analyze the number of parsimony- informative characters. To
check for gene-tree incongruence, I compared the PJ consensus gene trees (i.e. sequence
alignments 2 and 3).
The BI phylogenetic analyses were performed with MrBayes v.3.2.1 (Huelsenbeck &
Ronquist, 2001; Ronquist & Huelsenbeck, 2003). PartitionFinder (Guindon et al., 2010;
Lanfear et al., 2012; Lanfear et al., 2016) were utilized to set the priors for the analysis.
Settings were manually adjusted where branch lengths was set to linked, models of evolution
only compatible for MrBayes were selected, and the Bayesian information criterion (BIC)
was used for model selection. Data blocks were set for mtSSU, ITS1, 5.8S and ITS2. Schemes
search was set to greedy. In MrBayes, the Markov Chain Monte Carlo (MCMC) was run with
four chains (one cold and three heated chains) for 30 million generations, saving trees every
1000th generation. To check if the chains had converged, the average standard deviation of
split frequencies (ASDSF) was controlled. The ASDSF should fall below 0.01. All the
generations prior to the point of when the chains converged were discarded as burn-in, and the
remaining trees were summarized as a 50% majority-rule consensus tree. The tree was
visualized in FigTree (Rambaut, 2009), and edited with Microsoft® Powerpoint 2016. Only
sequence alignment 4 was analyzed with Bayesian Inference.
2.5 Morphological investigations
Microscope investigations were carried out on microtome sections cut at 20-30 µm or on
squash preparations. The preparations were observed in distilled water, 10% KOH (K),
lactophenol cotton blue (LCB) and a modified Lugol’s solution (I), where water was replaced
by 50% lactic acid. Asci were studied in I after a few seconds pretreatment in K. Polarized
light was used for locating crystals. The spore measurements are given as (x min) – x̄ - SD – x̄
– x̄ + SD – (x max), where x̄ is the mean length and SD the standard deviation.
16
2.6 Thin Layer Chromatography (TLC)
All specimens that showed to belonged in the genera of interest were subjected to thin-layer
chromatography (TLC) using the standard methods of Culberson and Kristinsson (1970) and
Culberson (1972), modified by Menlove (1974) and Culberson and Johnson (1982). All three
solvent systems (A, B and C) were used to examine the lichen substances.
17
3 Results
3.1 Sequences and alignments
In total, 65 of the 96 DNA extracts rendered PCR products that were successfully sequenced,
and several only for partial regions; 23 whole region sequences and 36 split region sequences
of the mtSSU, 15 whole region and 21 split region sequences of the ITS. Sequences with low
sequence quality, or that failed to show a relation within the focus clade after preliminary
phylogenetic analysis of sequence alignment 1, were removed. Of 59 newly produced mtSSU
sequences, 15 were included in the final analyses. Of 36 successful ITS sequences, 11 were
included in the final analyses. The success rate was generally lower for older specimens of all
species, and most of the DNA from the tropical specimens that were over 10 years of age did
not amplify. Five mtSSU and 21 ITS sequences were downloaded from GenBank. The four
analyzed sequence alignments (mtSSU alignment 1, mtSSU alignment 2, ITS alignment 3,
and concatenated alignment 4) were 1527, 998, 610, and 1608 bp long, with 683, 259, 244,
and 503 parsimony-informative characters (in-group, only), respectively.
PartitionFinder found three partitions in the concatenated sequence alignment 4 based on the
four pre-determined data blocks (see results chapter above), namely mtSSU, ITS1 & ITS2,
and 5.8s. The estimated best-fit nucleotide substitution models based on BIC were GTR+G
for mtSSU, SYM+G for ITS1 & 2, and K80+1 for 5.8s.
3.2 Phylogenetic analyses
The preliminary parsimony jackknife analyses showed congruent gene trees from mtSSU
alignment 2 and ITS alignment 3 (See Fig. S1: A and B), but resolved to various extents (ITS
being the more resolved).
In the BI-analysis, the ASDSF had fallen to 0.0043 at termination (30 million generations),
and the first 7500 trees (25%) were discarded as burn-in, removing all trees with an
ASDSF > 0.01 as recommended in the MrBayes manual. The remaining trees were
summarized into a Bayesian 50% majority-rule consensus tree, presented in Fig. 2. The
preliminary PJ phylogeny is mainly consistent with the BI phylogeny (see Fig. S2), with the
18
exception of a branch collapse of the 1433 Phyllopsora sorediata accession, in the BI-tree
(Fig. 2: Clade C).
The phylogenetic result (Fig. 2.) presents a well-supported topology that is highly resolved.
The branch support (PP/JK) is of varying degree, with the terminal branches having higher
support than the backbone. From the phylogenetic results, we can see that clade A has
received high branch support from the molecular analyses (1/ ≤ 85, PP/JK). Sister to clade A
is a not supported group of Bacidia. Clade A splits into two well-supported subclades (both 1/
≤ 85), hereby referred to as clade B and clade C.
Clade B includes 12 accessions of members tentatively determined to Phyllopsora, Physcidia
and Sporacestra. Basal in this clade, we find 395 Physcidia sp. as a highly supported (1/ ≤ 85)
sister to what is hereby called clade B’. Clade B’ contains 11 accessions of Sporacestra and
Phyllopsora, with high support (1/ ≤ 85). Both Sporacestra and Phyllopsora are paraphyletic,
based on the topology in clade B’. However, this is merely an artifact of preliminary naming
of the collections during fieldwork, where new collections have been named Sporacestra sp.
rather than Phyllopsora sp.
Clade C includes 31 accessions of species currently included in Bacidia, Bacidiopsora and
Phyllopsora. Three accessions from Thailand, tentatively determined by Einar Timdal as
Bacidiopsora sp. 1, are the most basal group in clade B with high branch support (≤ 0.95/71).
Bacidiopsora sp. 1 did not group together with the other members of Bacidiopsora, making
the genus polyphyletic.
Three accessions of Phyllopsora sorediata from Thailand group together with Bacidia
laurocerasi ssp. laurocerasi and Bacidia biatorina, but with low support (Fig. 2: clade C). In
the PJ phylogeny, P. sorediata group together with higher support (JK = 75; See Fig. 2: clade
C; Fig. S2: clade C). The topological placement of P. sorediata separated from P. borbonica
and P. pertexta (Fig. 2: clade B’), makes Phyllopsora polyphyletic in clade A.
As indicated above, Bacidia is polyphyletic and splits into two subclades. One of the
subclades lumps into clade C, and includes the type species of Bacidia (B. rosella). Nested
into Bacidia in clade C, we find the Bacidiopsora group, highly supported as monophyletic
(1/81). This monophyletic group includes eight accessions, both Neotropical (Venezuela and
19
Ecuador) and Pantropical (South Africa). See the discussion for more detailed information
about the phylogeny.
20
Spo
race
stra
0.0
5
Ba
cid
iasc
hw
ein
itzi
i
12
85
Sp
ora
cest
rasp
.1 M
alay
sia
56
17
Sp
ora
cest
rasp
.2B
razi
l
54
83
Lu
ecki
ng
iap
oly
spo
ra
51
1 P
hyl
lop
sora
bo
rbo
nic
aLa
Réu
nio
n H
T
56
19
Sp
ora
cest
rasp
. 2 B
razi
l
10
08
Ph
yllo
pso
raso
red
iata
Thai
lan
d
TT
Ba
cid
iaro
sella
Ba
cid
iaill
ud
ens
39
5 P
hys
cid
iasp
.C
ub
a
10
07
Ph
yllo
pso
raso
red
iata
Thai
lan
d
TT
14
28
Ba
cid
iop
sora
sp. 1
Thai
lan
d
48
05
Ba
cid
iop
sora
mic
rop
hyl
lina
Ve
nez
uel
a
56
14
Spo
race
stra
sp.2
Bra
zil
Ba
cid
iah
emip
olia
Ba
cid
iasu
ffu
sa
Ba
cid
iop
sora
squ
am
ulo
sula
SE3
14
Ecu
ado
r
10
15
Ba
cid
iop
sora
sp.1
Th
aila
nd
Ba
cid
iafr
axi
nea
56
32
Sp
ora
cest
rasp
. 2 B
razi
l
60
78
Ba
cid
iop
sora
sp. 2
So
uth
Afr
ica
Ba
cid
iasu
bin
com
pta
2Ba
cid
iasc
op
ulic
ola
14
33
Ph
yllo
pso
raso
red
iata
So
uth
Afr
ica
Ba
cid
iain
com
pta
56
18
Sp
ora
cest
rasp
. 2 B
razi
l
Ba
cid
iaa
uer
swa
ldii
Ba
cid
iala
uro
cera
siss
p.
lau
roce
rasi
60
74
Ba
cid
iop
sora
sp. 3
Sou
th A
fric
a
60
73
Ba
cid
iasp
.
Ba
cid
iaca
liga
ns
2
10
14
Ba
cid
iop
sora
sp.1
Thai
lan
d
Ba
cid
iab
ag
liett
oa
na
Ba
cid
iaa
bsi
sten
s
Ba
cid
iad
iffr
act
a
10
40
Ph
yllo
pso
rap
erte
xta
Cu
ba
60
75
Ba
cid
iop
sora
sp. 3
So
uth
Afr
ica
57
61
Ba
cid
iaw
ellin
gto
nii
Ba
cid
iasu
bin
com
pta
1
56
12
Sp
ora
cest
ra s
p.2
Bra
zil
60
79
Ba
cid
iop
sora
sp.2
So
uth
Afr
ica
Ba
cid
iab
iato
rin
a
Ba
cid
iave
rmif
era
2B
aci
dia
verm
ifer
a1
60
87
Ph
yllo
pso
ra p
erte
xta
Bra
zil
Ba
cid
ialu
tesc
ens
Ba
cid
iaru
bel
la
Ba
cid
iab
eckh
au
sii
42
7 P
hyl
lop
sora
bo
rbo
nic
aIn
do
nes
ia
Ba
cid
iasi
pm
an
ii Ba
cid
iap
oly
chro
a
48
06
Ba
cid
iop
sora
mic
rop
hyl
lina
Ve
nez
uel
a
60
37
Ba
cid
iop
sora
sp. 2
So
uth
Afr
ica
Ba
cid
iasp
.1
Ba
cid
iaa
rceu
tin
a
Ba
cid
iaca
liga
ns
1B
aci
dia
sp.
2
85
81
50
61
84
67
98
0,9
3
84
0,9
9
0,9
9
90
71
62
84
Ba
cid
ia s
. str
Fig
. 2:
The
Bay
esia
n5
0 %
maj
ori
tyru
leco
nse
nsu
s p
hylo
gra
mb
ased
on
57
acc
essi
ons
and
a 1
60
8 b
asep
air
long
alig
nm
ent,
fr
om
one
nucl
ear
(IT
S)
and
one
mit
och
ond
rial
(SS
U)
mar
ker
. It
consi
tso
fac
cess
ions
fro
m B
aci
dia
, B
aci
dio
pso
ra, P
hyl
lop
sora
and
Sp
ora
cest
ra.
Lu
ecki
ng
iap
oly
spo
rais
cho
sen
as o
utg
roup
. P
ost
erio
rp
rob
abil
itie
s(P
P)
is r
epo
rted
abo
ve
bra
nch
esan
d
par
sim
ony
jack
knif
e(J
K)
bra
nch
sup
po
rt u
nd
ernea
th.
Sup
po
rt v
alues
that
are
bel
ow
0.9
/50
(P
P/J
K)
are
no
t re
po
rted
.
ind
icat
es1≤ 8
5 n
od
e su
pp
ort
,
ind
ikat
esPP ≤ 0.95. Species
nam
esto
the
right
of
the
colo
ure
db
ars
ind
icat
eth
ecl
assi
fica
tio
nas
sup
po
rt h
erei
n.
Let
ters
to
the
left
in t
he
bac
kb
one
rep
rese
nts
clad
esin
tro
duce
din
the
Res
ult
sch
apte
r(c
lad
eA
, cl
ade
B, cl
adeB’ and
clad
eC
). H
T =
ho
loty
pe,
TT
= «
top
oty
pe» (
sam
e lo
cali
ty,
sam
e d
ate
as h
olo
typ
e).
Bra
nch
len
gth
sco
rres
po
nd
sto
num
ber
of
sub
stit
uti
ons
per
sit
e, a
ssum
ing
the
foll
ow
ing
sub
stit
ion
mo
del
s:
GT
R+
G f
or
mtS
SU
, S
YM
+G
fo
r IT
S1
& I
TS
2an
d K
80
+1
fo
r 5
.8s.
Bar
rep
rese
nts
0.0
5 %
chan
ge.
S. I
sid
iata
sp. n
ov.
S.
lon
gis
po
rasp
. n
ov.
S. p
ert
ext
a c
om
b.
no
v.
S. s
tra
min
ea
sp.
no
v.
B.
cora
llin
asp
. n
ov.
B.
sora
life
ran
om
. no
v.
B.
mic
rop
hyl
lan
om
. n
ov.
B.
kari
eg
ae
sp.
no
v
A
B
C
B’
21
3.3 Morphological investigations
Most of the Bacidia accessions were borrowed from GenBank, and no morphological or
anatomical investigations have been carried out on those specimens. Comparing the
morphology of the specimens in clade B (Fig. 2) clearly showed that the morphology of
Physcidia sp. differed from clade B’ by having a subfoliose thallus. The single collection of
this species was sterile, thus unsuitable for investigations on apothecial anatomy. Clade B’
contained five phenotypes, based on 26 specimens. All members shared the character of
having a well-developed brownish black prothallus with granules forming the thallus and long
non-septate ascospores, and differed mainly in the morphology of the apothecia.
Investigations on Bacidiopsora sp. 1 and the Bacidiopsora group in clade C was based on
four and eight specimens, respectively. They both shared the character of having a dark
prothallus and acicular, distinctly septate ascospores. They differed in that the Bacidiopsora
sp. 1 had a granulose thallus with coralloid isidia, whereas the Bacidiopsora group had a
squamulose thallus. Also, Bacidiopsora sp. 1 contained crystals in the excipulum only, while
the Bacidiopsora group contained crystals in the hypothecium as well. The Bacidiopsora
subgroup contained three phenotypes, differing in the presence of apothecia and the shape of
the squamules. The morphological investigations on Phyllopsora sorediata were based on
seven specimens and showed that the species possess a white to grey prothallus with an
areolate or subsquamulose thallus forming soralia. Further results from the morphological
investigation are elaborated in the Discussion chapter and presented in the Taxonomy chapter
below.
3.4 TLC
The TLC results revealed that eight lichen substances were present in clade A (Fig. 2). Most
of Bacidia was borrowed from GenBank, and no chemical investigations have been carried
out on those specimens. The specimens in clade B’ showed no trace of any lichen substances;
in contrast to the sister accession 395 Physcidia sp. which contained atranorin, zeorin and two
unknown compounds. All Phyllopsora sorediata specimens contained both atranorin and
divaricatic acid. Specimens in Bacidiopsora sp. 1 and the B. squamulosula group both showed
major occurrences of hyperhomosekikaic acid and/or homosekikaic acid. I was unable to
clearly distinguish between the two substances, although the former has a slightly higher Rf
value in solvent system B. These will hereafter be referred to as hyperhomosekikaic acid
22
Extr. Species/chemistry none atr div hhsek/hsek unk1 unk2 xan zeo fat
395 Physcidia sp.
1285 Sporacestra sp. 1
427 Phyllopsora pertexta
511 Phyllopsora borbonica
1040 Phyllopsora pertexta
6087 Phyllopsora pertexta
5632 Sporacestra sp. 2
5614 Sporacestra sp. 2
5619 Sporacestra sp. 2
5612 Sporacestra sp. 2
5617 Sporacestra sp. 2
5618 Sporacestra sp. 2
1014 Bacidiopsora sp. 1
1015 Bacidiopsora sp. 1
1428 Bacidiopsora sp. 1
1007 Phyllopsora sorediata
1008 Phyllopsora sorediata
1428 Phyllopsora sorediata
4806 Bacidiopsora microphyllina
4805 Bacidiopsora microphyllina
6078 Bacidiospora sp. 2
6079 Bacidiospora sp. 2
6037 Bacidiospora sp. 2
6074 Bacidiopsora sp. 3
6075 Bacidiopsora sp. 3
508 Bacidiopsora squamulosula
.
x
x
x
x
x
x
x
x
x
x
x
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
M
M
M
.
.
.
.
.
.
.
.
M
.
.
.
.
.
.
.
.
.
.
.
.
.
.
M
M
M
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
M
M
M
.
.
.
M
M
M
M
M
M
M
M
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
M
M
M
.
.
.
.
.
.
.
.
.
.
.
Table 4:Lichen substances detected in the focal g roup.
Abbrevations: Extr. = DNA extraction number, atr = atranorin, div = divaricatic acid, hhsek/hsek = hyperho-
mosekikaic acid/homosekikaic acid, unkn = unknown lichen substance, xan = unidentified xanthone,
zeo = zeorin, fat = fatty acid. M = Major occurrence.
M
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
M
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
M
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
and/or homosekikaic acid. Bacidiopsora sp.1 also contained an unidentified xanthone. An
overview over the lichen substances detected in the focal group is listed in Table 4.
23
4 Discussion
In this study, I assess the phylogenetic relationships in a poorly known group of tropical
lichens, namely clade A of the Ramalinaceae; see Fig. 1. Although the large genus Bacidia
has been extensively studied using both traditional and molecular methods (Czarnota &
Coppins, 2007; Ekman & Nordin, 1993; Lendemer et al., 2016; Llop & Gómez-Bolea, 1999),
there are only very few studies on the much smaller and presumably closely related genus
Bacidiopsora (A. Aptroot, 2002; Kalb, 1988, 2004; Kalb & Elix, 1995), of which none
include DNA sequence data. The possible resurrection of the genus Sporacestra as stated by
Ekman (1996) has been largely overlooked in the past (e.g. Lumbsch & Huhndorf, 2010;
Lücking et al., 2016), and no DNA sequences exist in GenBank under this or synonymous
names. Thus, this is the first molecular systematic study that includes members of the genera
Bacidiopsora, Phyllopsora and Sporacestra. The aim has been to examine the following
hypotheses: (1) Sporacestra represents a monophyletic genus; (2) Phyllopsora borbonica is
synonymous with P. pertexta; (3) Bacidiopsora sp. 1 represents a new genus; (4) Phyllopsora
sorediata represents a new genus; (5) Bacidiopsora is monophyletic, and; (6) Bacidiopsora is
not nested within Bacidia.
A large component of the biodiversity of lichenized fungi is overlooked and underestimated
partly due to the inconspicuous nature of small crustose lichens (Hodkinson & Lendemer,
2012). Many groups of lichenized fungi are taxonomically challenging due to few informative
phenotypic characters and a high level of homoplasy (Buschbom & Mueller, 2004; Gueidan et
al., 2007; Pino-Bodas et al., 2011). An example of this is the usage of thallus architecture as a
guide for delimiting lichen genera (Muggia et al., 2011), as exemplified by the typical
Phyllopsora growth form, i.e. having a squamulose thallus on a tightly woven prothallus. This
character seems to have evolved multiple times in the tropical habitat in the Ramalinaceae,
being present in at least Bacidiopsora, Eschatogonia, Krogia, Phyllopsora, and Physcidia (E.
Timdal, pers. comm.). Moreover, since the current knowledge of the phenotypic variation in
the focal genera is poor, my preliminary species determination in the field proved to be
challenging. Therefore, using an integrative approach to taxonomy as recommended by e.g.
Dayrat (2005) and Lumbsch & Leavitt (2011) , has been crucial in this study.
24
My phylogenetic results, based on two DNA markers and an expanded taxon sampling,
corroborates a highly supported clade in the Ramalinaceae that consists of several species
currently included in Bacidia, Bacidiopsora, Phyllopsora, and Sporacestra (Fig. 2: clade A).
The phylogenetic hypothesis is generally well supported, both in the deeper (the oldest
speciation events) but mostly in more terminal parts (recent speciation events; Fig. 2). The
Bayesian majority-rule consensus topology (Fig. 2: clade A) corroborates the preliminary
unpublished study of Bendiksby & Timdal (Fig. 1: clade A). Moreover, the status of Bacidia
as being polyphyletic also corroborates Ekman (2001), where largely the same species of
Bacidia were included. In the following section, I will discuss the members of clade B’ and
clade C (Fig. 2) in light of morphological (including anatomical), chemical, and molecular
results. Further, I will also propose taxonomic changes when there is support from multiple
sources of evidence.
4.1 Clade B’: Sporacestra and Phyllopsora
The molecular phylogeny shows that there is a clade, namely clade B’ in Fig. 2, that is highly
supported (1/≤85). The clade comprises species preliminary designated to a resurrected
Sporacestra, together with four accessions of P. pertexta and P. borbonica. The
circumscription of Phyllopsora is under revision (Kistenich et al., in prep.), and the core of
the genus, including the type species, P. breviuscula, belongs outside clade A (Bendiksby &
Timdal, unpubl.; Fig 1). The apparent paraphyly of both genera in clade B’ is, as earlier
mentioned, merely an artefact of preliminary naming. As already stated, P. pertexta is
synonymous with the type species of Sporacestra (Biatora prasina), this being the basis of
the resurrection of Sporacestra. Thus, hypothesis (1) Sporacestra is monophyletic, can be
accepted based on this phylogeny.
It became apparent during the fieldwork that separating what might represent members of
Sporacestra from some members of Phyllopsora was almost impossible. Based on the 11
accessions representing clade B’, with 15 additional specimens, the characters delimitating
this genus was investigated. The results showed that the main delimiting character separating
Sporacestra from Bacidia, Bacidiopsora, and Phyllopsora is the morphology of the
ascospores, a character impossible to use in the field without microscopy. In more detail,
Sporacestra differs from Bacidia and Bacidiopsora in having long, acicular, non-septate to
pseudoseptate ascospores. Moreover, the genus differs from Bacidiopsora by not containing
25
any lichen substances. Sporacestra is morphologically similar to Phyllopsora, and the only
reliable evidence for separating members in the two genera appears to be with molecular
analyses. The genus Aciculopsora, described from Costa Rica (Aptroot et al., 2006), is also
morphologically similar to Sporacestra in having a crustose thallus and acicular ascospores.
This genus, however, belongs outside clade A (S. Kistenich, pers. comm.).
Basal in clade B’ we find Sporacestra sp. 1 (Fig. 2) as a highly supported sister to the other
accessions in clade B’. It is apparent from the phylogeny that this species is genetically
separated from the other members of Sporacestra, illustrated with a long branch.
Morphological investigations on the sequenced and four additional specimens revealed that
this species is separated from the other members of clade B’ by the presence of isidia. With
both the molecular and morphological evidence, I decide to describe this as a new species, S.
isidiata to be included in the resurrected Sporacestra (see the Taxonomy chapter below)
Further in clade B’, we can see from the phylogeny that P. borbonica is represented by two
accessions (427 and 511, the holotype) that are genetically separated. The accession 427 P.
borbonica is a sister to 511 P. borbonica, P. pertexta and Sporacestra. sp. 2, indicating that it
may represent an undescribed species. Alternatively, it might indicate geographic distance
and lack of gene flow. Results of a sequence identity matrix between 427 P. borbonica and
the accessions of 511 P. borbonica revealed that the similarity was at 50%. Closer
morphological (including anatomy) examinations on the sequenced and 11 additional
specimens affirmed distinct differences, including dull yellow, plane to concave apothecia in
427 P. borbonica, in contrast to brown, plane to convex apothecia in P. borbonica. On the
basis of both the molecular data, combined with the morphological, I therefore conclude that
427 P. borbonica constitutes a new undescribed species, S. longispora, to be described in the
Taxonomy chapter below.
Nested into clade B’, we find a subclade with P. pertexta and P. borbonica. Phyllopsora
pertexta was described from the Neotropics (Cuba) some 150 years ago (Nylander, 1863),
while P. borbonica was described much later from the Pantropics (La Réunion and Mauritius;
Timdal & Krog, 2001). Timdal & Krog (2001) did not discuss or compare P. borbonica with
P. pertexta. Later, however, Timdal (2011) commented on the possible synonymy of the two
species, and indicated that the color of the hypothecium is the only character distinguishing
them. In this study, I was not able to separate the two species in the coloration of the
hypothecium, nor in any other character. Comparing the DNA sequence of the two species, a
26
sequence identity matrix revealed that the sequences are 97% similar, compared to the
similarity of P. borbonica to the other sequences in clade B’, which all fell under 50%. The
split between 511 P. borbonica (the holotype) from the two accessions of P. pertexta (from
Cuba and Brazil) could be due to the fact that the accession is only represented by mtSSUA
and ITS2. Another explanation for the split could be due to the geographical distance between
the accessions, 511 P. borbonica being Pantropical, where the two accessions of P. pertexta
are Neotropical. On the basis of this, it would not be appropriate to keep them described as
two separate species. I therefore consider P. borbonica and P. pertexta to be synonymous and
the new combination S. pertexta is formally made in the Taxonomy chapter below. Moreover,
with this I accept hypothesis (2) P. pertexta is synonymous with P. borbonica.
Sister to S. pertexta in clade B’ (Fig. 2) is a subclade that consists of six accessions of
Sporacestra sp. 2 from two different localities in Brazil (see Table 1). The accessions are
highly homogeneous molecularly, and the genetic distinctness and internal homogeneity of
this group support this as a good species. Alternatively, this could represent a new genus,
based on the high degree of genetic divergence portrayed by the long branch. However, since
this clade share most characters with the rest of clade B’ (Fig. 2: clade B’), as the character of
having long acicular spores, there is no basis of describing this as a new genus, when clade B’
strongly appears as a natural group. The subclade is distinguished from most species currently
included in Sporacestra by the plane to convex, dull yellow apothecia. On account of the
molecular, morphological, and anatomical results, I regard this as a new species to be
described as S. straminea (see the Taxonomy chapter below).
The molecular data, combined with morphology (including anatomy) and chemistry, revealed
that the 11 accessions in the phylogeny (Fig. 2: clade B’) represents four species to be
included in a monophyletic, resurrected Sporacestra. Clade B’ is highly supported in every
internal branch, except for a lack of support in the S. pertexta and S. straminea group
(>0.9/50). As both the S. pertexta and S. straminea clade receive high support, this might
indicate a lack of taxon sampling and more undescribed species may belong in this clade. On
the other hand, an alternative polytomy with S. longispora, S. pertexta and S. straminea may
represent three closely evolving species and a further support on the monophyly of
Sporacestra.
27
4.2 Clade C: Bacidia, Bacidiopsora and Phyllopsora
My phylogenetic result highly supports clade C (Fig. 2; 1/≤85), consisting of Bacidia,
Phyllopsora sorediata and members currently included in Bacidiopsora as a sister to clade B.
Bacidia is a large genus with high morphological and anatomical variation. The generic
description I follow was made by Ekman (1996) on Bacidia species from North America, and
there has never been made a modern revision of all Bacidia species worldwide. In this
phylogeny (Fig. 2) we can see that Bacidia is polyphyletic with its type species (Bacidia
rubella) present together with other members in clade C. Many members of Bacidia fall
outside clade A. The same is shown in Ekman (2001), where he defines the species present in
this clade C as Bacidia sensu stricto, a definition I hereby adopt. Several members of Bacidia
sensu lato has recently been studied and moved to other genera (Kalb et al., 2000; Lücking et
al., 2001).
Basal in clade C, we find three accessions tentatively determined as Bacidiopsora by Timdal
(1014, 1015 and 1428 Bacidiopsora sp. 1 from Thailand, see Fig. 2) with high PP support
(=1). The determination was based upon the presence of a dark prothallus, a squamulose
thallus, pluriseptate ascospores, and the content of hyperhomosekikaic and/or homosekikaic
acid. The molecular phylogenetic result tells a different story, placing Bacidiopsora sp. 1
outside the rest of Bacidiopsora, indicating that these Bacidiopsora type characters are a
product of convergent evolution (cfr. Muggia et al., 2011).
The three accessions of P. sorediata (1007, 1008 and 1433, all “topotypes” = collected on the
same date, same locality as holotype: Fig. 2: clade C) are not supported as monophyletic, and
there seems to be a geographic component to it; the two accessions from Thailand form a
supported group, whereas 1433 from South Africa falls out in a polytomy that also includes a
clade with Bacidia laurocerasi ssp. laurocerasi and Bacidia biatorina. The branch collapse of
1428 P.sorediata in the BI-phylogeny (See fig. 2; Appendix Fig. S2) is probably due to the
fact that the accession is only represented by mtSSUB. This species were originally described
and placed in Triclinum by Aptroot (2007), based on the prothallus, squamulose thallus and
the presence of atranorin, and divaricatic acid. Timdal (2011) transferred it to Phyllopsora on
the basis of his synonymization of Triclinum with Phyllopsora. The preliminary phylogeny
(Fig. 1; Bendiksby & Timdal, unpubl.) shows that P. sorediata does not belong in
28
Phyllopsora. Ongoing studies by Kistenich and co-workers show that it does not belong in
Triclinum either (S. Kistenich, pers. comm.).
My DNA sequence phylogeny places a second clade of Bacidiopsora accessions (the one
including the type species of the genus, B. squamulosula) nested within Bacidia s. str., thus
rejecting hypothesis (5) Bacidiopsora is monophyletic and (6) Bacidiopsora is not nested into
Bacidia. In addition to the type species, the Bacidiopsora clade appear to hold three distinct
genetic lineages (Fig. 2: clade C, B. microphyllina, sp. 2, and sp. 3), which gains support also
from morphological and anatomical investigations. Three accessions (6037, 6078 and 6079),
referred to as Bacidiopsora sp. 2, has the same morphology and anatomy as B. microphyllina,
and I conclude that they represent the same species. The split between Bacidiopsora
microphyllina and sp. 2 in the phylogeny could be explained by the geographical distance and
hence lack of gene flow between the populations, the former being Neotropical (Venezuela),
the latter Pantropical (South Africa). The sister clade to Bacidopsora microphyllina (incl. sp.
2) consists of Bacidiopsora squamulosula and two accessions (6074 and 6075) referred to as
Bacidiopsora sp. 3. The latter species differs from all other species currently included in
Bacidiopsora by having squamules dissolving into granular soredia (Fig. 5, Taxonomy
chapter below). The taxon sampling of Bacidiopsora is still not satisfying, as my attempts to
sequence B. orizabana, B. psorina, B. silvicola, and B. tenuisecta, all failed. Most of the
specimens (except B. tenuisecta from BR) were borrowed from herbarium Klaus Kalb, now in
WIS, that was collected in the tropics from 1980–2002. The collections may therefore be too
old for molecular work, due to degradation of DNA (cfr. Gueidan et al., 2016). Alternatively,
it could be that the methods used for DNA extraction in this study are not well-suited for old
specimens and low DNA concentration, and an adjustment of the extraction procedure may be
needed.
As I see it, there are two alternatives for the taxonomic treatment of Bacidiopsora sp. 1,
Bacidiopsora, and P. sorediata. (1) Lumping the three groups into Bacidia s. str., thus making
clade C one big, anatomically and morphologically variable, but monophyletic genus. In that
case, the concept of the genus Bacidia s. str. will be extended to include species with a well-
developed squamulose thallus, pseudoseptate ascospores, and with the secondary compounds
divaricatic acid, hyperhomosekikaic acid, and homosekikaic acid. (2) Splitting the clade,
putting the strongest weight on morphology, describing Bacidiopsora sp. 1 and P. sorediata
as to two new genera. Thus, keeping a monophyletic Bacidiopsora within clade C, making
29
Bacidia s.str. paraphyletic. However, as discussed earlier in this chapter, thallus morphology
may not be a good character at the genus level, merely a rather easily evolving adaption to
ecological conditions (Gueidan et al., 2007). Moreover, the presence or absence of lichen
substances is due to the expression of the genotype, and may be a product of the lichen’s
ecological adaption (Brodo, 1986). This can be seen in the sekikaic acid-complex, which is
widely distributed throughout the Ramalinaceae, present in Bacidiopsora, Eschatogonia,
Phyllopsora, Physcidia, and Ramalina (Kalb, 2004; Krog & James, 1977; Swinscow & Krog,
1988; Timdal, 2008). This shows that the secondary chemistry may not be a good delimiting
character at the genus level. Now, with the addition of molecular data to infer on the
evolutionary relationship, this should be treated as first priority in delimiting genera
(Lumbsch & Leavitt, 2011). Choosing alternative (2) could make the taxonomy complicated,
and may be misleading about character distribution and evolution. Furthermore, I believe that
it would be premature to split the clades when Bacidia s. lat. is in such an unstable state
regarding its circumscription, due to the lack of molecular data for the majority of the species.
Therefore, I conclude with taxonomical alternative (1), which is to lump Bacidiopsora sp. 1,
Bacidiopsora, and P. sorediata into Bacidia s. str., which in turn leads to rejection of both
hypotheses (3) and (4), i.e. that Bacidiopsora sp. 1 and P. sorediata represents two new
genera. Moreover, I will describe Bacidiopsora sp. 1 and Bacidiopsora sp. 3 as two new
species to be included in Bacidia, namely, Bacidia corallina and Bacidia kariegae. New
names are needed for B. microphyllina and P. sorediata as the epithets are already used in
Bacidia (see the Taxonomy chapter, below).
4.3 Conclusion and final remarks
This study investigated the phylogenetic relationships within the tropical genera Bacidia,
Bacidiopsora and Sporacestra (clade A) in the family Ramalinaceae. It proved to be
challenging to obtain quality DNA sequence data from this group of tropical lichens. The
quality of the molecular data was thoroughly gone through by manually editing and excluding
sequences with low quality, to ensure the best results from the analyses. The results in this
thesis show that many phenotypic characters in the Ramalinaceae are a product of convergent
evolution, and this emphasizes the importance of molecular systematics. The integrative
approach, combining traditional taxonomy (morphology, anatomy, and chemistry) and
molecular methods, led me to the conclusions that Sporacestra is a monophyletic genus and
30
that all species in clade C should be lumped into Bacidia s. str. A consequence of this is that I
describe five new species, synonymize one species name (Phyllopsora borbonica),
synonymize one genus name (Bacidiopsora), resurrect one genus name (Sporacestra), make
one new combination, and two new names in Bacidia s. str. (see the Taxonomy chapter,
below).
During this two-year period of working with the thesis, several complications arose and
became clear along the way. First of all, working with in the tropical habitat is in itself a
challenging task, due to the warm, humid conditions in the dense vegetation. To be able to
perform such a task, you are completely dependent on local researchers, not only to guide and
plan the logistics, but also to obtain research permits. International agreements, e.g. the
Nagoya protocol, has made it challenging to collect outside Norway, especially in Brazil,
where everything collected has to be registered in a local herbarium and from there sent to
Norway as duplicates or loans. This procedure can often take up to several months, and it
does not help that genomic DNA of crustose, corticolous lichens seems to degrade quite fast
after specimen desiccation (Gueidan et al., 2016). Moreover, species (even genus)
identification in the field may also be difficult, especially when the genera in question have
few diagnostic characters. Most of my fieldwork collections were excluded from the dataset
after preliminary analysis of sequence alignment 1 (Table 1, indicated with *), because they
fell outside clade A or the sequence quality was too low to be included.
4.4 Further studies
This study is only scratching the surface of the undescribed species diversity in the Tropics. If
I had the opportunity to prolong this study, I would have gone back in the laboratory
optimizing procedures with the aim to obtain both genetic markers for all included accessions.
Moreover, DNA sequences of the four remaining species of Bacidiopsora (B. orizabana, B.
psorina, B. silvicola, and B. tenuisecta) have to be successfully obtained and placed in a
molecular phylogeny before further conclusions can be made. Future studies should also
include additional genetic markers, e.g. the protein-coding, nuclear RPB1 and MCM7
markers, to increase the chance of the phylogeny to reflect the true evolutionary history and
relationship of these clades. These markers have been shown to be useful in species
delimitation on lichens (e.g. Divakar et al., 2017; Schmitt et al., 2012). I would also
recommend trying the procedure of using FTA cards on freshly collected tropical specimens,
31
as recommended by Gueidan et al. (2016). In this procedure, DNA from tropical collections is
extracted directly from specimens in the field (by the use of Polybutylene terephthalate
[PBT]) and stored on FTA cards, which bind and preserves the DNA. Lastly, and most
importantly, Bacidia, Bacidiopsora and Sporacestra should be placed into a Ramalinaceae
phylogeny, to ensure broad and extensive taxon sampling. This can provide further
understanding of these rather inconspicuous tropical species.
32
5 Taxonomy
Sporacestra A. Massal.
in Atti Reale Ist. Veneto Sci. Lett. Arti., ser 3, 5: 264 (1860). Type: Biatora prasina Mont. &
Tuck. [non (Fr.) Fr.] [= Sporacestra pertexta].
Prothallus present, white when young, becoming brownish black. Thallus crustose, granulose,
containing unicellular green algae; cortex one or two cell layers thick, composed of rather
thin-walled hyphae with rounded lumina. — Apothecia biatorine, yellow to brown; proper
excipulum colorless to pale brown in inner part, colorless to dark brown in the rim, composed
of radiating, conglutinated, rather thick-walled hyphae with shortly cylindrical lumina;
hypothecium not distinctly delimited from exipulum, colorless to reddish or dark brown,
chondroid, composed of irregularly oriented, thick-walled hyphae with cylindrical lumina;
hymenium colorless, with amyloid gelatin, 70–90 µm high; paraphyses conglutinated,
straight, simple or rarely branched, c. 5 µm wide, with a slightly swollen, colorless or faintly
brown, apical cell; ascus narrowly clavate, with a well-developed, amyloid tholus usually
with an ocular chamber; ascospores acicular, straight, parallel in the ascus, simple or
irregularly pseudoseptate, colorless. — Pycnidia unknown. Chemistry: No lichen substances
(by TLC).
33
Sporacestra isidiata Stapnes & Timdal, sp. nov. [MB XXXXX]
Diagnosis: Prothallus thick, brownish black. Thallus with isidia patchily distributed on the
thallus; isidia cylindrical, simple, c. 0.06 wide and 0.6mm long, pale brownish green.
Typus: Malaysia, Malaysian Borneo, Sabah, Maliau Basin, 4.7476N, 116.9675E, pristine
lowland dipterocarp forest, 2012, P. Wolseley, H. Thüs, & C. Vairappan M.3.08.oQ.2
(BORH, holotypus! [TLC: No lichen substances detected; GenBank: XXXXXXXX]).
(Fig. 3A)
Prothallus thick, white when young, soon becoming brownish black. Thallus effuse, crustose,
formed by small (up to 0.1 mm wide), adnate, isodiametric granules which are discrete to
adjoined peripherally and soon forming a more or less continuous crust; granules pale green,
glabrous, pubescent along the margin; isidia present, often patchily distributed, originating
one per granule, cylindrical, c. 0.06 mm wide, up to 0.6 mm long, simple, pale brownish
green, glabrous; upper cortex composed of 1–2 layers of thin walled hyphae with rounded
cells, 5–10 µm thick; algal layer filling the inner part of the granules; cortex and algal layer
not containing crystals. — Apothecia and pycnidia not seen.
Distribution and ecology. Malaysian Borneo in the Maliau Basin. Crustose on tree bark in
pristine lowland dipterocarp forest.
Notes. Sporacestra isidiata differs from all other species in Sporacestra by the presence of
isidia. Apothecia have not been seen in this species. The organization and morphology of the
external and internal structures is similar to the other species included in the genus.
Etymology. Isidata, named after the presence of isidia.
Additional species examined: Malaysia: Malaysian Borneo, Sabah, Maliau Basin, 4.7476N, 116. 9676E,
pristine lowland dipterocarp forest, 2012, P. Wolseley, H. Thüs & C. Vairappan, M-132, M3-0I-2, & M3-03-2,
(all BORH).
34
Sporacestra longispora Stapnes, sp. nov. [MB XXXXX]
Diagnosis: Apothecia rounded to flexuose, often conglomerate, plane to moderately concave,
yellowish brown; margin paler than the disc; ascospores acicular to filiform, 26–104 ×
c.2 µm.
Typus: Indonesia, Kalimantan Selan, Hulu Tabalong, 135’05’S, 11531’27’E, old logged (c.
1983) dipterocarp forest, 630 m alt., 3 April 2000, P. Wolseley T13 LQ (BM 001104062 –
holotypus! [TLC: No lichen substances detected; GenBank: XXXXXXXX]).
(Fig. 3B)
Prothallus thin to thick, white when young, later becoming brownish black. Thallus effuse,
crustose, formed by small (up to 0.1 mm wide), adnate, isodiametric granules which are
discrete to adjoined peripherally and soon form a more or less continuous crust; granules pale
green and glabrous, sometimes slightly pubescent along the margin; isidia and soredia
lacking; upper cortex composed of 1–2 layers of thin-walled hyphae with rounded cells, 5–
10 µm thick; algal layer filling the inner part of the granules; cortex and algal layer not
containing crystals. — Apothecia common, up to 1.2 mm diam., rounded or with a flexuose
margin, simple or conglomerate, plane to moderately concave, yellowish brown; margin
sometimes slightly pubescent when young, paler than the disc, rarely blackening on the
underside; excipulum colorless throughout; hypothecium pale brown; epithecium colorless;
no crystals in the apothecia; ascospores acicular to filiform, simple or with up to 3
pseudosepta, (26)–41–60–79–(104) × c. 2 µm (n=50). — Pycnidia not seen.
Distribution and ecology. Japan, Malaysia, and Papua New Guinea. Crustose on tree bark in
pristine lowland primary forest at 160–1500 m alt.
Notes. Sporacestra longispora is similar to S. straminea by having yellowish brown apothecia
but differs in having plane to concave apothecia, rather than plane to convex. It differs from S.
pertexta by having yellowish brown, not reddish to purple brown apothecia; furthermore, it
differs from both in forming longer, partly filiform ascospores
Etymology. Longispora, named after its delimiting character of long spores.
Additional species examined: Japan: Yaeyama Islands, Iriomote-jima island along the trail in the mountains
along the Urauchi river from 500 m n of the southernmost starting point 6 km NW of Ohara village to the small
35
camping site between two streams 2.5 km NNE of the starting point (7.5 km NNW of Ohara village), Taketomi-
cho, Yaegama-gun, 2419’N, 12351’E, 160-220 m alt., 1995, G. Thor 13197, 13216 (UPS-L-392055, 392056).
Malaysia: Malaysian Borneo, Sabah, Maliau Basin, 4.6972N, 116.906E, pristine lowland dipterocarp forest,
2012, P. Wolseley, H. Thüs & C. Vairappan, M.87 (BORH). Papua New Guinea: Morobe, ridge SE of
Bouwao, SW of Law, 1470’E, 70’S, primary forest, on tree bark, 1500 m alt., 1965, A.C. Jeremy 1499 (BM
000577330). Bulolo, Mt. Selk, 18 km from Bulolo on the way to the Lake, upphill, W of the road, 14640’E,
715’S, secondary forest on tree bark, 750-850 m alt., 1965, D.J. Hil 12290 (BM 000921683).
36
37
Sporacestra pertexta (Nyl.) Stapnes & Timdal, comb. nov. [MB
XXXXX].
Lecidea pertexta Nyl. in Ann. Sci. Nat., Bot., ser. 4, 19: 347 (1863) Psorella pertexta
(Nyl.) Müll. Arg. in Bot. Jahrb. Syst. 23: 297 (1897) Phyllopsora pertexta (Nyl.) Swinscow
& Krog in Lichenologist 13: 244 (1989). Type: Cuba,”in ins. Cuba”, C. Wright. s.n. (H-NYL
17344, holotype [TLC: No lichen substances, according to Timdal 2011]).
= Biatora prasina Tuck. & Mont. in Ann. Sci. Nat., Bot., ser. 4, 8: 296 (1857), nom. illeg.
[non (Fr.) Fr. in Stirpes Agri Femsionensis: 38 (1826)] Biatora prasinata Tuck. in Syn. N.
Amer. Lich. 2: 41 (1888). Type: Venezuela, s. loc. A. Fendler s.n. (H-NYL 21859a, isotype
[image!]) Bacidia prasinata (Tuck.) Coppins in Bull. Br. Mus. Nat. Hist., Botany 11: 17-
214 (1983).
= Phyllopsora borbonica Timdal & Krog in Mycotaxon 77: 68 (2001). Type: La Réunion,
along road towards Plain d’Affoches, above Bras Citron, at point where road meets track.
2057’S, 5525’E, alt 1220 m, on tree trunk in forest. 1996-09-26 H. Krog & E. Timdal
RE8/12 (O-L-797, holotype! [TLC: No lichen substances, according to Timdal & Krog 2001;
GenBank: XXXXXXXX]).
(Fig. 4A)
Prothallus medium thick, brownish black, light brown to white when young. Thallus effuse,
crustose, formed by small (up to 0.1 mm wide), adnate, isodiametric granules which are
discrete to adjoined peripherally and form a more or less continuous crust centrally; granules
pale green and glabrous, often pubescent along the margin (at least when discrete); isidia and
soredia lacking; upper cortex composed of 1–2 layers of thin-walled hyphae with rounded
cells, 5–15 µm thick; algal layer filling the inner part of the granules; cortex and algal layer
not containing crystals. — Apothecia abundant, up to 2 mm diam., rounded or with a flexuose
margin, simple, plane to moderately convex (plane when young), reddish to purple brown;
margin sometimes slightly pubescent when young, paler than the disc, blackening on the
underside; excipulum pale brown to colorless in inner part, pale brown to dark reddish brown
in the rim; hypothecium dark brown to reddish brown in the upper part, paler in the lower
part; epithecium colorless; no crystals in apothecia; reddish brown pigment in excipulum and
38
hypothecium K+ purple; ascospores acicular, simple, with up to 3 pseudosepta, (17)–25–32–
39–(53) × c. 2 µm (n=39). — Pycnidia not seen.
Distribution and ecology. I have verified specimens from: Brazil, Cuba, Mauritius, Papua
New Guinea, La Réunion, and Venezuela. It is also reported from Samoa (Müller, 1897). The
species is found on the bark in humid forests and humid woodlands at 530–1255 m alt.
Notes. Sporacestra longispora is morphologically similar, but differs in having lighter brown,
more concave apothecia with a margin lighter colored than the disc. Sporacestra straminea
differs from S. pertexta in having dull yellow apothecia.
Additional specimens examined: Brazil: Rio Abajo, 2014, M. Cáceres, M. 20131, 20134 (ISE). Cuba: Pinar del
Rio, La Palma, Magoto El Pan de Guajaibón, 2247’N, 8322’W, 705 m alt., 2006 S. Pérez-Ortega (hb. Pérez-
Ortega). Mauritius: Macchabee Forest (0.5–1 km ESE of Macchabee Kiosk), 2024’S, 5726’E, 600 m alt.,
2006, H. Krog & E. Timdal,MAU13/08 (O-L-21341). Rivière Noire District, Black River Gorges National Park;
along trail to Piton de la Petite Rivière Noire, 20.42133N, 57.41947E, 630-700 m alt., 2016, Diederich, P., (hb.
Diederich 18432, 18454). Papua New Guinea: Morobe: Herzog Mts, Wago, ridge above village, 710’S,
14640’E, primary forest, 1100 m alt., 1965, A.C. Jeremy 4902 (BM 000577345). Seychelles: Mahé: Mare aux
Cochons, on a Cinnamomum species, 1994, K. Beaver, (hb. Seaward 114397). La Réunion: Fôret de Bébour,
along road towards Takamaka, by small bridge WSW of the end of the road, 2105’S, 5535’E, 1255 m alt.,
1996, H. Krog & E. Timdal RE30/11(O-L-74322). Le Grand Etang, along the trail from the road to the lake,
2105’S, 5539’E, 530-540 m alt. 1996, H. Krog & E. Timdal RE30/11 (O-L-74323).
39
Sporacestra straminea Stapnes, sp. nov. [MB XXXXX]
Diagnosis: Apothecia common, up to 1.5 mm diam., mostly rounded, simple, dull yellow;
margin paler than the disc, sometimes blackening on the underside.
Typus : Brazil, Pará, Melgaço, Floresta Nacional de Caxiuanã, Estação Científica Ferreira
Penna, 142.850’S, 5127.207’W (WGS84), on tree trunk in tropical rainforest, 30–50 m alt.,
2015-03-14, S. Kistenich & E. Timdal SK1-097 (O-L-201106, holotype! [TLC: No lichen
substances detected; GenBank: XXXXXXXX]).
(Fig. 4B)
Prothallus thin to thick, white when young, later becoming brownish black. Thallus effuse,
crustose, formed by small (up to 0.1 mm wide), adnate, isodiametric granules which are
discrete to adjoined peripherally and soon form a more or less continuous crust; granules pale
green and glabrous, sometimes slightly pubescent along the margin; isidia and soredia
lacking; upper cortex composed of 1-2 layers of thin walled hyphae with rounded cells, 5-
10 µm thick; algal layer filling the inner part of the granules; cortex and algal layer not
containing crystals. — Apothecia common, up to 1.5 mm diam., mostly rounded, simple,
plane to moderately convex, dull yellow; margin sometimes slightly pubescent when young,
paler than the disc, sometimes slightly blackening on the underside; excipulum colorless
throughout; hypothecium pale brown; epithecium colorless; no crystals in apothecia;
ascospores acicular, simple or with up to 3 pseudosepta, (38)–47–53–58–(65) × c. 2 µm
(n=40). — Pycnidia not seen.
Distribution and ecology. Brazil. On tree trunks in Amazonian rainforests.
Notes. The species resembles both S. longispora and S. pertexta in most of its external and
internal morphological characters, but differs from both in the appearance of the apothecia.
The color of the apothecia, dull yellow, can resemble apothecia in S. longispora, but the disc
is convex rather than concave. The species differs from S. pertexta in the color of the
apothecia, whereas S. straminea has dull yellow apothecia, S. pertexta has reddish to purple
brown.
Etymology. Straminea, named after its dull yellow apothecia.
40
Additional specimens examined: Brazil: Pará, Melgaço, Floresta Nacional de Caxiuanã,
Estação Científica Ferreira Penna, 142.847’S to 144.332’S, 5127.200’W to 5127.667’W,
on tree trunk in tropical rainforest, 30-50 m alt., 2015-03 Kistenich, S. & Timdal, E., SK1-011,
-024, -039,-092 (O-L-201020,-201033,-201048,-201101). Pará, Tailândia, Fazenda
Agroecológica São Roque, 304.501’S, 4901.0881W, on tree trunk in tropical rainforest, 50
m alt., 2015-11 Dahl et al. SK1-316 (O-L-202596).
41
42
Appendix: Bacidia De Not.
In Giorn. Bot. Ital. 2: 189 (1846). Type: Bacidia rosella (Pers.) De Not.
Bacidia corallina Stapnes & Timdal, sp. nov. [MB XXXXX]
Diagnosis: Similar to the Bacidiopsora group of Bacidia but with coralloid isidia.
Typus: Thailand, Uthau Thani. Site: Kapu Kapiang station, Khlong Plou, 9916’E, 1539’N,
in dry evergreen forest, 500 m alt., 1993-02, Aguirre, James & Wolseley 2478a (BM
000749861, holotype! [TLC: Hyperhomosekikaic acid and/or homossekikaic acid and
unidentified xanthone detected; GenBank: XXXXXXX]).
(Fig. 5A)
Prothallus thick, black. Thallus corticated, formed by small, adnate, isodiametric granules up
to 0.3 mm diam., remaining discrete; granules pale greenish gray (herbaria material); isidia
abundant, attached marginally to the granules, coralloid, c 0.03 mm wide, up to 0.4 mm long;
upper cortex up to 10 µm thick, composed of 2–3 layers of thick-walled hyphae with rounded
lumina, not containing crystals; algal layer filling the inner part of the granules, densely
incrusted by crystals refracting polarized light and dissolving in K; — Apothecia common, up
to 1.2 mm diam., rounded, plane to slightly convex, yellowish pale brown, blackening on the
underside, not pubescent; proper excipulum composed of radiating, conglutinated, thick-
walled hyphae with broadly cylindrical to ellipsoid lumina, reddish brown in the rim (K+
purpur), pale brown in the inner part, containing crystals refracting polarized light;
hypothecium yellowish brown, not distinctly delimited from exipulum, chondroid, composed
of irregularly oriented, thick-walled hyphae with cylindrical lumina, not containing crystals;
hymenium colorless (with amyloid gelatin), 100–120 µm high; paraphyses conglutinated,
straight, simple or rarely branched, c. 4 µm wide, with a slightly swollen, colorless apical cell;
ascus narrowly clavate, with a well-developed tholus; tholus amyloid in upper part, lacking
ocular chamber and axial mass; ascospores acicular, straight or slightly curved, parallel in the
ascus, ± 7-septate, colorless, (39)–49–55–62–(65) × c. 2 µm (n=15). — Pycnidia unknown.
Chemistry. Hyperhomosekikaic and/or homosekikaic acid and xanthones.
43
Notes. The species is similar to members of the Bacidiopsora group of Bacidia, but differs in
containing additional xanthones, having a granulose thallus with coralloid isidia, yellowish
brown apothecia, and crystals only in the excipulum, not in the hypothecium. The excipulum
is thinner than in Bacidiopsora and is composed of hyphae with shorter, more broadly
cylindrical to ellipsoid lumina.
Distribution and ecology. Thailand. Crustose on bark in dry evergreen forest at 500 m alt.
Etymology. Corallina, named after the coralloid isidia.
Additional specimens examined: Thailand: Uthai Thani. Site: Kapou Kapiang station, Khlong Plou, 9916’E,
1539’N, in dry evergreen forest, 500 m alt., 1993-02, Aguirre, James & Wolseley 4277e (BM 001031544).
Khao Nang Rum Viewpoint track 2.2, 9918’E, 1529’N, in dry everegreen forest, 370-460 m alt., Aguirre,
James & Wolseley 2479a, 2715 (BM 000749844, 000749853).
Bacidia kariegae Stapnes, sp. nov. [MB XXXXX]
Diagnosis: Similar to the Bacidiopsora group of Bacidia but with granular soralia.
Typus: South Africa, Eastern Cape, Western District: 11 km NNW of Kenton-on-Sea,
3335.36’S, 2637.31’E, on tree trunk in low, dry forest, 160 m alt., 2015-09, S. Rui & E.
Timdal13875 (O-L-202868, holotype! [TLC: Hyperhomosekikaic and/or homosekikaic acid
detected; GenBank: XXXXXXXX])
(Fig. 5B)
Prothallus, thick, black. Thallus corticated, formed by small, ascending, irregularly imbricate,
isodiametric squamules up to 0.5 mm diam.; squamules dull, pale green, soon breaking in to
effuse, granular soralia; upper cortex up to 15 µm thick, composed of 2–3 layers of thick-
walled hyphae with rounded lumina, not containing crystals; algal layer up to 20 µm thick;
medulla densely incrusted by crystals refracting polarized light and dissolving in K. —
Apothecia and pycnidia not seen.
Chemistry. Hyperhomosekikaic acid and/or homosekikaic acid.
Notes. The species resembles Bacidia microphylla and differs mainly in forming granular
soralia.
44
Distribution and ecology. South Africa, known from the type locality only. On tree trunk in
low dry forest.
Etymology. Kariegae, from the type locality in the Kariega Game Reserve.
Additional specimens examined: South Africa, Eastern Cape, Western District, 11 km NNW of Kenton-on-Sea,
3335.35’S, 2637.23’E, on tree trunk in low, dry forest, 160 m alt., 2015-09, S. Rui & E. Timdal13874 (O-L-
202867).
Bacidia microphylla Stapnes, nom. nov. [MB XXXXX]
Bacidiopsora microphyllina Kalb in Biblioth. Lichenol. 88: 304 (2004) [non Bacidia
microphyllina (Tuck.) Riddle, Mycologia 15: 80 (1923)].
Note. The species is here reported new to South America.
Additional specimens examined: South Africa: Mpumalanga, Ehlanzeni District, Buffelkloof Nature Reserve,
2516.12’S, 3030.97’E, on tree in mist forest, 1700 m alt., 2014-10, J. Burrows & E. Timdal, 14204, 14214,
14219 (O-L196747, 196757, 196762). Venezuela: Capital District, Parque Nacional Waraira Repano,
1032.827’N, 6652.629’W, on tree trunk in tropical moist Forest, 1800 m alt., 2015-11, Dahl et al., SK1-204,
SK1-205 (O-L-202484, 202485).
Bacidia soralifera Stapnes & Timdal, nom. nov. [MB XXXXX]
Triclinum sorediatum Aptroot & Sparrius in Aptroot et al., Fungal Diversity 24: 130 (2007)
Phyllopsora sorediata (Aptroot & Sparrius) Timdal in Lichenologist 40: 337 (2008) [non
Bacidia sorediata Lendemer & R.C. Harris, Bryologist 119: 167 (2016)].
(Fig. 5C)
Prothallus white to pale grey, poorly developed. Thallus effuse, crustose to subsquamulose,
formed by medium sized (up to 0.3 mm wide) plane to weakly convex, adnate or partly
ascending, isodiametric areolae or squamules; areolae and squamules scattered when young,
later becoming contiguose or ascending and irregularily imbricate, greyish green (according
to Aptroot et al. 2007), not pubescent; soralia punctiform when young, later irregular and
45
often confluent, granular, white; upper cortex up to 15 µm, composed of more or less
irregularly arranged, thick-walled hyphae with angular lumina, containing crystals refracting
polarized light; algal layer filling the inner part, containing smaller crystals; photobiont green
unicellular algae, up 10 µm diam. — Apothecia common, up to 1 mm diam., mostly rounded,
simple, weakly concave to moderately concave, dull yellow to brownish yellow; margin thick
when young, later becoming more indistinct, paler than the disc, not blackening on the
underside; excipulum colorless with crystals; hypothecium colorless; epithecium colorless
with a few scattered crystals; ascospores acicular, straight or slightly curved, simple or with
irregular pseudosepta, (18)–22–27–33–(44) × c. 2 µm (n=40). — Pycnidia not seen.
Chemistry. Atranorin, divaricatic acid and two unknowns.
Notes. A full description based on my investigation of material collected at the same locality
and date as the holotype is given here because the original description of Aptroot et al. (2007)
seems to be based on mixed material (i.e., the specimen containing fumarprotocetraric acid).
The species is here reported as new to Africa.
Distribution and ecology. Known from South Africa and Thailand, crustose on bark in dry
dipterocarp forest at 460–640 m alt.
Additional specimens examined: South Africa: Eastern cape, Grootrivier (1km N of Natures Valley not far from
the bridge), 3358’S. 2334’E, on trunk of Podocarpus falcatus, 10 m., Anders Nordin, 4642 (UPS L.92604)
Thailand: Uthai Thani. Site: Kapou Kapiang station, khlong Plou, 9911’E to 9916’E, 1530’N to 1539’N, on
bark in dry evergreen forest, 500 m alt., 1993-03, Aguirre,, James & Wolseley, 2806, 2854, 2877, 2906 (BM
000749892, 000763640, 000749760, 000749761). Khao Nang Rum Viewpoint track 205, 9918’E, 1529’N, on
bark in dry dipterocarp forest, 460-640 m alt., 1992-01, B. Aguriee-Hudson & P.A. Wolseley, 1397, 3948 (BM
000749859, 000749765).
46
47
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51
A.
B.
Fig
. S
1:
two
par
sim
ony
jack
knif
e(P
J) c
onse
nsu
s phylo
gen
ies.
(A
)Is
bas
edon
34 a
cces
sions
and a
998 b
asep
airs
long
alig
nm
ent
(seq
uen
ceal
ignm
ent
2)
from
one
mit
och
ondri
al(S
SU
) m
arker
. (B
)Is
bas
edon
42 a
cces
ions
and a
610 b
asep
air
long
alig
nm
ent
(seq
uen
ceal
ignm
ent
3)
from
one
nucl
ear
(IT
S)
mar
ker
. B
oth
phylo
gen
ies
consi
sts
of
acce
ssio
ns
from
Baci
dia
, B
aci
dio
pso
ra, P
hyl
lopso
ra a
nd S
pora
cest
ra.Ja
ckknif
e(J
K)
bra
nch
support
are
report
edab
ove
bra
nch
es.
54
83
Lu
ecki
ng
ia p
oly
spo
raB
aci
dia
inco
mp
ta S
E11
7B
aci
dia
illu
den
s SE
30
63
95
Ph
ysci
dia
sp.
12
85
Sp
ora
cest
rasp
. 1M
alay
sia
42
7 P
hyl
lop
sora
bo
rbo
nic
aIn
do
nes
ia5
11
Ph
yllo
pso
ra b
orb
on
ica
La
Réu
nio
n1
04
0P
hyl
lop
sora
per
text
aC
ub
a5
63
2 S
po
race
stra
sp. 2
Bra
zil
56
14
Spo
race
stra
sp. 2
Bra
zil
56
19
Sp
ora
cest
rasp
. 2 B
razi
l5
61
2 S
po
race
stra
sp. 2
Bra
zil
56
17
Sp
ora
cest
rasp
. 2 B
razi
l5
61
8 S
po
race
stra
sp. 2
Bra
zil
10
14
Ba
cid
iop
sora
sp. 1
Th
aila
nd
10
15
Ba
cid
iop
sora
sp. 1
Th
aila
nd
14
28
Ba
cid
iop
sora
sp. 1
Th
aila
nd
10
07
Ph
yllo
pso
ra s
ore
dia
ta T
hai
lan
d1
00
8 P
hyl
lop
sora
so
red
iata
Th
aila
nd
14
33
Ph
yllo
pso
ra s
ore
dia
taSo
uth
Afr
ica
Ba
cid
ia a
bsi
sten
s SE
93
Ba
cid
ia s
ipm
an
ii JQ
79
68
32
Ba
cid
ia a
rceu
tin
a S
E23
Ba
cid
ia s
uff
usa
KJ7
66
355
57
61
Ba
cid
ia w
ellin
gto
nii
60
73
Ba
cid
ia s
p.
Ba
cid
ia s
chw
ein
itzi
i DQ
97
299
8B
aci
dia
ro
sella
SE5
0B
aci
dia
ru
bel
la D
Q9
86
808
48
06
Ba
cid
iop
sora
mic
rop
hyl
lina
Ven
ezu
ela
60
74
Ba
cid
iop
sora
sp. 3
Sou
th A
fric
a6
07
5 B
aci
dio
pso
rasp
. 3So
uth
Afr
ica
60
78
Ba
cid
iop
sora
sp. 2
So
uth
Afr
ica
60
79
Ba
cid
iop
sora
sp. 2
Sou
th A
fric
a
50
8 B
acid
iop
sora
sq
uam
ulo
sula
60
37
Ba
cid
iop
sora
sp. 2
Sou
th A
fric
a
75
73
71
87
87
99
10
0
71
95
81
99
10
0
99
81
64
73
70
98
92
54
71
96
83
10
0
Ton
inia
ca
nd
ida
Ba
cid
ia in
com
pta
Ba
cid
ia il
lud
ens
Ba
cid
ia b
ag
liett
oa
na
Ba
cid
ia a
uer
swa
ldii
Ba
cid
ia s
ub
inco
mp
ta1
Ba
cid
ia s
ub
inco
mp
ta2
Ba
cid
iasp
. 1B
aci
dia
sp. 2
Ba
cid
ia c
alig
an
s 1
Ba
cid
ia c
alig
an
s 1
Ba
cid
ia h
emip
olia
Ba
cid
ia b
eckh
au
sii
Ba
cid
ia v
erm
ifer
a1
Ba
cid
ia v
erm
ifer
a2 42
7P
hyl
lop
sora
bo
rbo
nic
a I
nd
on
esia
51
1 P
hyl
lop
sora
bo
rbo
nic
aLa
Réu
nio
n1
04
0P
hyl
lop
sora
per
text
aC
ub
a6
08
7 P
hyl
lop
sora
per
text
aB
razi
l5
63
2Sp
ora
cest
ra s
p. 2
Bra
zil
56
14
Sp
ora
cest
rasp
. 2 B
razi
l5
61
9 S
po
race
stra
sp
. 2 B
razi
l5
61
2 S
po
race
stra
sp. 2
Bra
zil
56
17
Sp
ora
cest
rasp
. 2 B
razi
l5
61
8 S
po
race
stra
sp. 2
Bra
zil
10
14
Ba
cid
iop
sora
sp. 1
Th
aila
nd
Ba
cid
ia lu
tesc
ens
Ba
cid
ia la
uro
cera
si s
sp. l
au
roce
rasi
Ba
cid
ia b
iato
rin
a
Ba
cid
ia a
bsi
sten
sB
aci
dia
sco
pu
lico
laB
aci
dia
arc
euti
na
Ba
cid
ia s
uff
usa
Ba
cid
ia s
chw
ein
itzi
i Ba
cid
ia f
raxi
nea
Ba
cid
ia p
oly
chro
a
Ba
cid
ia d
iffr
act
a
Ba
cid
ia r
ose
lla Ba
cid
ia r
ub
ella
48
06
Ba
cid
iop
sora
mic
rop
hyl
lina
Ven
ezu
ela
48
05
Ba
cid
iop
sora
mic
rop
hyl
lina
Ven
ezu
ela
60
74
Ba
cid
iop
sora
sp. 3
Sou
th A
fric
a6
07
5 B
aci
dio
pso
rasp
. 3 S
ou
th A
fric
a
Ba
cid
iop
sora
sq
ua
mu
losu
la
70
53
10
0
10
0
10
0
10
0
10
0
99
99
84
10
0
99
10
0
63
99
10
0
10
0
63
83
75
71
10
0
64
60
10
0
97
90
75
53
10
0
Appendix
Figure S1: PJ phylogeny of sequence alignment 2
(A) and 3 (B).
52
54
83
Lu
ecki
ng
ia p
oly
spo
ra
Ba
cid
ia a
uer
swa
ldii
Ba
cid
ia in
com
pta
Ba
cid
ia h
emip
olia
Ba
cid
ia b
eckh
au
sii
Ba
cid
ia il
lud
ens
Ba
cid
ia s
ub
inco
mp
ta1
Ba
cid
ia s
ub
inco
mp
ta2
Ba
cid
iasp
. 1B
aci
dia
sp
. 2B
aci
dia
ca
liga
ns
1B
aci
dia
ca
liga
ns
2
Ba
cid
ia v
erm
ifer
a1
Ba
cid
ia v
erm
ifer
a2
Ba
cid
ia b
ag
liett
oa
na
10
14
Ba
cid
iop
sora
sp. 1
Th
aila
nd
42
7 P
hyl
lop
sora
bo
rbo
nic
aIn
do
nes
ia5
11
Ph
yllo
pso
ra b
orb
on
ica
La R
éun
ion
10
40
Ph
yllo
pso
ra p
erte
xta
Cu
ba
60
87
Ph
yllo
pso
rap
erte
xta
Bra
zil
56
32
Sp
ora
cest
rasp
. 2 B
razi
l5
61
4 S
po
race
stra
sp. 2
Bra
zil
56
19
Sp
ora
cest
rasp
. 2 B
razi
l5
61
8 S
po
race
stra
sp. 2
Bra
zil
56
12
Sp
ora
cest
rasp
. 2B
razi
l5
61
7 S
po
race
stra
sp. 2
Bra
zil
Ba
cid
ia la
uro
cera
si s
sp. l
au
roce
rasi
Ba
cid
ia b
iato
rin
a
Ba
cid
ia a
bsi
sten
s
57
61
Ba
cid
ia w
ellin
gto
nii
Ba
cid
ia s
chw
ein
itzi
i
Ba
cid
ia a
rceu
tin
aB
aci
dia
sco
pu
lico
la
Ba
cid
ia p
oly
chro
aB
aci
dia
dif
fra
cta
Ba
cid
ia s
uff
usa
Ba
cid
ia r
ose
llaB
aci
dia
ru
bel
laB
aci
dia
fra
xin
ea
48
06
Ba
cid
iop
sora
mic
rop
hyl
lina
Ven
ezu
ela
48
05
Ba
cid
iop
sora
mic
rop
hyl
lina
Ven
ezu
ela
Bac
idio
pso
ra s
qu
amu
losu
la S
E31
4
Ba
cid
ia lu
tesc
ens
60
74
Ba
cid
iop
sora
sp. 3
So
uth
Afr
ica
60
75
Ba
cid
iop
sora
sp. 3
So
uth
Afr
ica
60
78
Ba
cid
iop
sora
sp. 2
So
uth
Afr
ica
60
37
Ba
cid
iop
sora
sp. 2
So
uth
Afr
ica
60
79
Ba
cid
iop
sora
sp. 2
So
uth
Afr
ica
Ph
ysci
dia
sp. 3
95
Cu
ba
12
85
Sp
ora
cest
rasp
. 1 M
alay
sia
Ba
cid
ia s
ipm
an
ii
10
15
Ba
cid
iop
sora
sp. 1
Thai
lan
d1
42
8 B
aci
dio
pso
rasp
. 1 T
hai
lan
d
Ph
yllo
pso
ra s
ore
dia
ta 1
00
7 T
hai
lan
dP
hyl
lop
sora
so
red
iata
10
08
Th
aila
nd
Ph
yllo
pso
ra s
ore
dia
ta 1
43
3 S
ou
thA
fric
a
60
73
Ba
cid
ia s
p.
90
68
96
97
96
10
0
87
61
99
88
99
62
10
0
75
85
86
84
67
71
91
10
0
81
50
84
94
54
98
95
63
98
90
Fig.
S2
: Th
e p
arsi
mo
ny
jack
kn
ife
(PJ)
co
nse
nsu
s p
hylo
gen
yb
ased
on
57
acc
essi
on
san
d a
16
08
bas
epai
rslo
ng
alig
nm
ent
fro
m o
ne
nu
clea
r(I
TS
) an
d o
ne
mit
och
on
dri
al(S
SU
) m
arker
. It
co
nsi
sts
of
acce
ssio
ns
fro
m B
aci
dia
, B
aci
dio
pso
ra, P
hyl
lop
sora
an
d S
po
race
stra
.Ja
ckkn
ife
(JK
) b
ran
chsu
pp
ort
are
rep
ort
edab
ove
bra
nch
es.
Figure S2: PJ phylogeny of sequence alignment 4